Protease variants and compositions

ABSTRACT

The present invention relates to enzymes produced by mutating the genes for a number of subtilases and expressing the mutated genes in suitable hosts are presented. The enzymes exhibit improved autoproteolytic stability in comparison to their wild type parent enzymes.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.08/963,851 filed Nov. 4, 1997, and claims priority of Danish applicationnos. 1235/96, 1240/96 and 0284/97 filed Nov. 4, 1996, Nov. 5, 1996 andMar. 14, 1997, respectively, the contents of which are fullyincorporated herein by reference.

TECHNICAL FIELD

[0002] This invention relates to novel mutant protease enzymes or enzymevariants useful in formulating detergent compositions and exhibitingincreased autoproteolytic stability; cleaning and detergent compositionscontaining said enzymes; mutated genes coding for the expression of saidenzymes when inserted into a suitable host cell or organism; and suchhost cells transformed therewith and capable of expressing said enzymevariants.

BACKGROUND OF THE INVENTION

[0003] In the detergent industry enzymes have for more than 30 yearsbeen implemented in washing formulations. Enzymes used in suchformulations comprise proteases, lipases, amylases, cellulases, as wellas other enzymes, or mixtures thereof. Commercially most importantenzymes are proteases.

[0004] Although proteases have been used in the detergent industry formore than 30 years, much remains unknown as to details of how theseenzymes interact with substrates and/or other substances present in e.g.detergent compositions. Some factors related to specific residues of theproteases and influencing certain properties, such as oxidative andthermal stability in general, of the proteases have been elucidated, butmuch remains to be found out. Also, it is still not exactly known whichphysical or chemical characteristics are responsible for a good washingperformance or stability of a protease in a specific detergentcomposition.

[0005] The currently used proteases have for the most part been found byisolating proteases from nature and testing them in detergentformulations.

[0006] At present at least the following proteases are known to becommercially available and many of them are marketed in large quantitiesin many countries of the world.

[0007] Subtilisin BPN′ or Novo (available from e.g. SIGMA, St. Louis,U.S.A.), and Subtilisin Carlsberg, ALCALASE® (NOVO NORDISK A/S)) andMAXATASE®(Genencor).

[0008] A Bacillus lentus subtilisin, subtilisin 309, marketed by NOVONORDISK A/S as SAVINASE®. A protein engineered variant of this enzyme ismarketed as DURAZYM®.

[0009] Enzymes closely resembling SAVINASE®, such as subtilisin PB92,MAXACAL® marketed by Genencor Inc. (a protein engineered variant of thisenzyme is marketed as MAXAPEM®), OPTICLEAN® marketed by SOLVAY et Cie.and PURAFECT® marketed by GENENCOR International.

[0010] A Bacillus lentus subtilisin, subtilisin 147, marketed by NOVONORDISK A/S as ESPERASE®;

[0011] An increasing number of commercially used proteases are proteinengineered variants of naturally occurring wild type proteases, e.g.DURAZYM® (Novo Nordisk A/S), RELASE® (Novo Nordisk A/S), MAXAPEM®(Gist-Brocades N.V.), PURAFECT® (Genencor International, Inc.).

[0012] Therefore, an object of the present invention, is to provideimproved protein engineered protease variants, especially for use in thedetergent industry.

[0013] Proteases

[0014] Enzymes cleaving the amide linkages in protein substrates areclassified as proteases, or (interchangeably) peptidases (see Walsh,1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, SanFrancisco, Chapter 3). Bacteria of the Bacillus species secrete twoextracellular types of protease, neutral proteases (ormetalloproteases), and alkaline proteases among which the most importantfunctionally is a serine endopeptidase and usually referred to assubtilisin.

[0015] Serine Proteases

[0016] A serine protease is an enzyme which catalyzes the hydrolysis ofpeptide bonds, and in which there is an essential serine residue at theactive site (White, Handler and Smith, 1973 “Principles ofBiochemistry,” Fifth Edition, McGraw-Hill Book Company, NY, pp.271-272).

[0017] The bacterial serine proteases have molecular weights in the20,000 to 45,000 Daltons range. They are inhibited bydiisopropylfluorophosphate. They hydrolyze simple terminal esters andare similar in activity to eukaryotic chymotrypsin, also a serineprotease. A more narrow term, alkaline protease, covering a sub-group,reflects the high pH optimum of some of the serine proteases, from pH9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41711-753).

[0018] Subtilases

[0019] A sub-group of the serine proteases tentatively designatedsubtilases has been proposed by Siezen et al., Protein Engng. 4 (1991)719-737. They are defined by homology analysis of more than 40 aminoacid sequences of serine proteases previously referred to assubtilisin-like proteases. A subtilisin was previously defined as aserine protease produced by Gram-positive bacteria or fungi, andaccording to Siezen et al. now is a subgroup of the subtilases. A widevariety of subtilisins have been identified, and the amino acid sequenceof a number of subtilisins have been determined. These include more thansix subtilisins from Bacillus strains, namely, subtilisin 168,subtilisin BPN′, subtilisin Carlsberg, subtilisin Y, subtilisinamylosacchariticus, and mesentericopeptidase (Kurihara et al. (1972) J.Biol. Chem. 247 5629-5631; Wells et al. (1983) Nucleic Acids Res. 117911-7925; Stahl and Ferrari (1984) J. Bacteriol. 159 811-819, Jacobs etal. (1985) Nucl. Acids Res. 13 8913-8926; Nedkov et al. (1985) Biol.Chem. Hoppe-Seyler 366 421-430, Svendsen et al. (1986) FEBS Lett. 196228-232), one subtilisin from an actinomycetales, thermitase fromThermoactinomyces vulgaris (Meloun et al. (1985) FEBS Lett. 198195-200), and one fungal subtilisin, proteinase K from Tritirachiumalbum (Jany and Mayer (1985) Biol. Chem. Hoppe-Seyler 366 584-492). forfurther reference Table I from Siezen et al. has been reproduced below.

[0020] Subtilisins are well-characterized physically and chemically. Inaddition to knowledge of the primary structure (amino acid sequence) ofthese enzymes, over 50 high resolution X-ray structures of subtilisinshave been determined which delineate the binding of substrate,transition state, products, at least three different proteaseinhibitors, and define the structural consequences for natural variation(Kraut (1977) Ann. Rev. Biochem. 46 331-358).

[0021] One subgroup of the subtilases, I-S1, comprises the “classical”subtilisins, such as subtilisin 168, subtilisin BPN′, subtilisinCarlsberg (ALCALASE®, NOVO NORDISK A/S), and subtilisin DY.

[0022] A further subgroup of the subtilases I-S2, is recognized bySiezen et al. (supra). Sub-group I-S2 proteases are described as highlyalkaline subtilisins and comprise enzymes such as subtilisin PB92(MAXACAL®, Gist-Brocades NV), subtilisin 309 (SAVINASE®, NOVO NORDISKA/S), subtilisin 147 (ESPERASE®, NOVO NORDISK A/S), and alkalineelastase YaB.

[0023] Random and site-directed mutations of the subtilase gene haveboth arisen from knowledge of the physical and chemical properties ofthe enzyme and contributed information relating to subtilase's catalyticactivity, substrate specificity, tertiary structure, etc. (Wells et al.(1987) Proc. Natl. Acad. Sci. U.S.A. 84; 1219-1223; Wells et al. (1986)Phil. Trans. R. Soc. Lond.A. 317 415-423; Hwang and Warshel (1987)Biochem. 26 2669-2673; Rao et al., (1987) Nature 328 551-554.

[0024] More recent publications covering this area are Carter et al.(1989) Proteins 6 240-248 relating to design of variants that cleave aspecific target sequence in a substrate (positions 24 and 64); Graycaret al. (1992) Annals of the New York Academy of Sciences 672 71-79discussing a number of previously published results; and Takagi (1993)Int. J. Biochem. 25 307-312 also reviewing previous results.

[0025] Especially site-directed mutagenesis of the subtilisin genes hasattracted much attention, and various mutations are described in thefollowing patent applications and patents:

[0026] Previously Characterized Protease Variants

[0027] Numerous references describe construction of Protease variants.In order to make it easier to get an overview of the overall prior artstatus, the references are ordered in two sections.

[0028] Section one deals with references describing the technologicalbackground and identification of Protease variants presently notbelieved to be related to the variants disclosed in the presentinvention. Those references are mainly included in order to summarizethe state of the art within the field of construction of proteasevariants for different purposes.

[0029] Section two deals with references describing identification ofProtease variants believed to be of some relevance to the variants ofthe present invention.

[0030] References Summarizing the State of the Art (Section 1):

[0031] EP 130756 (GENENTECH)(corresponding to U.S. Reissue Pat. No.34,606 (GENENCOR)) relating to site specific or randomly generatedmutations in “carbonyl hydrolases” and subsequent screening of themutated enzymes for various properties, such as k_(cat)/K_(m) ratio,pH-activity profile, and oxidation stability. This publication claimssite-specific mutation of subtilisins BPN′ in certain specifiedpositions, i.e. ⁻¹Tyr, ³²Asp, ¹⁵⁵Asn, ¹⁰⁴Tyr, ²²²Met, ¹⁶⁶Gly, ⁶⁴His,¹⁶⁹Gly, ¹⁸⁹Phe, ³³Ser, ²²¹Ser, ²¹⁷Tyr, ¹⁵⁶Glu or ¹⁵²Ala, to provide forenzymes exhibiting altered properties. Since these positions all exceptposition −1 were known to be involved in the functioning of the enzymeprior to the filing of the application, this application does notcontribute to solving the problem of deciding where to introducemutations in order to obtain enzymes with specific desired properties.

[0032] EP 214435 (HENKEL) relating to cloning and expression ofsubtilisin Carlsberg and two mutants thereof. In this application noreason for mutation of ¹⁵⁸Asp to ¹⁵⁸Ser and ¹⁶¹Ser to ¹⁶¹Asp isprovided.

[0033] In WO 87/04461 (AMGEN) it is proposed to reduce the number ofAsn-Gly sequences present in the parent enzyme in order to obtainmutated enzymes exhibiting improved pH and heat stabilities, in theapplication emphasis is put on removing, mutating, or modifying the¹⁰⁹Asn and the ²¹⁸Asn residues in subtilisin BPN′. No examples areprovided for any deletions or for modifying the Gly-residues.

[0034] WO 87/05050 (GENEX) discloses random mutation and subsequentscreening of a number of mutants of subtilisin BPN′ for improvedproperties. In the application mutations are described in positions²¹⁸Asn, ¹³¹Gly, ²⁵⁴Thr, ¹⁶⁶Gly, ¹¹⁶Ala, ¹⁸⁸Ser, ¹²⁶Leu, and ⁵³Ser.

[0035] EP 260105 (GENENCOR) describes modification of certain propertiesin enzymes containing a catalytic triad by selecting an amino acidresidue within about 15 Å from the catalytic triad and replace theselected amino acid residue with another residue. Enzymes of thesubtilase type described in the present specification are specificallymentioned as belonging to the class of enzymes containing a catalytictriad. In subtilisins positions 222 and 217 are indicated as preferredpositions for replacement.

[0036] Also, it has been shown by Thomas, Russell, and Fersht (1985)Nature 318 375-376 that exchange of ⁹⁹Asp into ⁹⁹Ser in subtilisin BPN′changes the pH dependency of the enzyme.

[0037] In a subsequent article (1987) J. Mol. Biol. 193 803-813, thesame authors also discuss the substitution of ¹⁵⁶Ser in place of ¹⁵⁶Glu.

[0038] Both these mutations are within a distance of about 15 Å from theactive ⁶⁴His.

[0039] In Nature 328 496-500 (1987) Russel and Fersht discuss theresults of their experiments and present rules for changing pH-activityprofiles by mutating an enzyme to obtain changes in surface charge.

[0040] WO 88/08028 (Genex) and WO 88/08033 (Amgen) both relate tomodifications of amino acid residues in the calcium binding sites ofsubtilisin BPN′. The enzyme is said to be stabilized by substitutingmore negatively charged residues for the original ones.

[0041] WO 95/27049 (SOLVAY S.A.) describes a subtilisin 309 typeprotease with the following mutations: N43R+N116R+N117R (BPN′ numbering.Data indicate the corresponding variant is having improved stability,compared to wildtype.

[0042] WO 95/30011, WO 95/30010, and WO 95/29979 (PROCTER & GAMBLECOMPANY) describe 6 regions, especially position 199-220 (BPN′numbering), in both Subtilisin BPN′ and subtilisin 309, which aredesigned to change (i.e. decrease) the adsorption of the enzyme tosurface-bound soils. It is suggested that decreased adsorption by anenzyme to a substrate results in better detergent cleaning performance.No data on any variants or specific detergent wash performance data areprovided.

[0043] Previously Characterized Protease Variants Having Some Relevanceto the Present Invention (Section Two):

[0044] In EP 251 446 (GENENCOR) it is described how homologyconsiderations at both primary and tertiary structural levels may beapplied to identify equivalent amino acid residues whether conserved ornot. It is claimed that this information together with the inventorsknowledge of the tertiary structure of subtilisin BPN′ brought theinventors to select a number of positions susceptible to mutation withan expectation of obtaining mutants with altered properties. Thepositions so identified are: ¹²⁴Met, ²²²Met, ¹⁰⁴Tyr, ¹⁵²Ala, ¹⁵⁶Glu,¹⁶⁶Gly, ¹⁶⁹Gly, ¹⁸⁹Phe, ²¹⁷Tyr. Also ¹⁵⁵Asn, ²¹Tyr, ²²Thr, ²⁴Ser, ³²Asp,³³Ser, ³⁶Asp, ⁴⁶Gly, ⁴⁸Ala, ⁴⁹Ser, ⁵⁰Met, ⁷⁷Asn, ⁸⁷Ser, ⁹⁴Lys, ⁹⁵Val,⁹⁶Leu, ¹⁰⁷Ile, ¹¹⁰Gly, ¹⁷⁰Lys, ¹⁷¹Tyr, ¹⁷²Pro, ¹⁹⁷Asp, ¹⁹⁹Met, ²⁰⁴Ser,²¹³Lys, and ²²¹Ser, which positions are identified as being expected toinfluence various properties of the enzyme. Also, a number of mutationsare exemplified to support these suggestions. In addition to singlemutations in these positions the inventors also performed a number ofmultiple mutations. Further the inventors identify ²¹⁵Gly, ⁶⁷His,¹²⁶Leu, ¹³⁵Leu, and amino acid residues within the segments 97-103,126-129, 213-215, and 152-172 as having interest, but mutations in anyof these positions are not exemplified.

[0045] Especially of interest for the purpose of the present inventionthe inventors of EP 251 446 suggest to substitute ¹⁷⁰Lys (in subtilisinBPN′, type I-S1), specifically they suggest to introduce Glu or Arg forthe original Lys. It appears that the Glu variant was produced and itwas found that it was highly susceptible to autolytic degradation (cf.pages 48, 121, 123 (Table XXI includes an obvious error, but indicates areduction in autolysis half-time from 86 to 13 minutes) and FIG. 32).

[0046] In WO 89/06279 (NOVO NORDISK A/S) position 170 is indicated asinteresting and it is suggested to replace the existing residue withTyr. However, no data are given in respect of such a variant. In WO91/00345 (NOVO NORDISK A/S) the same suggestion is made, and it is shownthat the Tyr variant of position 170 in subtilisin 309 (type I-S2)exhibits an improved wash performance in detergents at a pH of about 8(variant S003 in Tables III, IV, V, VI, VIII, X). The same substitutionin combination with other substitutions in other positions alsoindicates an improved wash performance (S004, S011-S014, S022-S024,S019, S020, S203, S225, S227 in the same Table and Table VII) all inaccordance with the generic concept of said application.

[0047] In EP 525 610 Al (SOLVAY) it is suggested to improve thestability of the enzyme (a type I-S2 subtilase closely related tosubtilisin PB92) towards ionic tensides by decreasing the hydrophobicityin certain surface regions thereof. It is suggested to substitute Ginfor the Arg in position 164 (170 if using BPN′ numbering). No variantscomprising this substitution are disclosed in the application.

[0048] In WO 94/02618 (GIST-BROCADES N.V.) a number of position 164 (170if using BPN′ numbering) variants of the I-S2 type subtilisin PB92 aredescribed. Examples are provided showing substitution of Met, Val, Tyr,and Ile for the original Arg. Wash performance testing in powderdetergents of the variants indicates a slight improvement. Especiallyfor the lie variant wash performance tests on cacao an improvement ofabout 20-30% is indicated. No stability data are provided.

[0049] In PCT/DK96/00207 (not yet published) a number of proteasevariants with improved wash performance and/or storage stability isdescribed.

[0050] It was found that subtilase variants with improved washperformance can by obtained by substituting one or more amino acidresidues situated in, or in the vicinity of a hydrophobic domain of theparent subtilase for an amino acid residue more hydrophobic than theoriginal residue, said hydrophobic domain comprising the residuescorresponding to residues I165, Y167, Y171 of BLS309 (in BASBPNnumbering), and said residues in the vicinity thereof comprisingresidues corresponding to the residues E136, G159, S164, R170, A194, andG195 of BLS309 (in BASBPN numbering) (PCT/DK96/00207).

[0051] U.S. Pat. No. 5,543,302 describes the identification of importantautoproteolytic cleavage site in different wild type proteases(MILEZYME®, SAVINASE®, and ESPERASE®).

[0052] The present invention focuses on specific protease variantsinfluencing the position of autoproteolytic cleavage sites in a group ofprotease variants described in our pending patent applicationPCT/DK96/00207. The variants of the present invention have surprisinglybeen found to exhibit an altered autoproteolytic degradation patterncompared to the autoproteolytic degradation pattern described forcorresponding wildtype proteases in U.S. Pat. No. 5,543,302.

SUMMARY OF THE INVENTION

[0053] It has now surprisingly been found that subtilase variants havingmodification(s) in one or more amino acid residues situated in positionscorresponding to residues P129, P131, E136, G159, S164, I165, Y167,R170, Y171 of BLS309 (in BASBPN numbering), have an alteredautoproteolytic degradation pattern, as compared to the correspondingwildtype. The primary alteration is an autolytic cleavage siteapparently located between residues 132-133 (in BASBPN numbering).

[0054] SAVINASE® is known to have a broad substrate specificity and willupon extensive incubation auto cleavage itself at numerous sites.However the initial 1-5 cleavage sites (primary sites) are of highestrelevance for industrial applications of the subtilase proteases.

[0055] Said autolytic cleavage site located between residues 132-133 ispresently believed to be a new primary autoproteolytic cleavage site.This is presently believed to be the first time that thisautoproteolytic cleavage site has been identified, as a primaryautoproteolytic cleavage site, in a subtilase protease enzyme. Forfurther details reference is made to a working example herein (videinfra).

[0056] Consequently in its first aspect, the present invention relatesto a subtilase enzyme variant characterized by being derived from aprecursor subtilase enzyme having an autoproteolytic split site betweenresidues 132 and 133 (in BASBPN numbering), which further has beenmodified by substitution, insertion or deletion at or in one or more ofthe residues situated in positions corresponding to residues 129, 130,131, 132, 133, 134, 135, 136 of BLS309 (in BASBPN numbering); wherebysaid variant exhibits increased autoproteolytic stability relative tosaid precursor subtilase enzyme.

[0057] In a second aspect the invention relates a subtilase enzymevariant characterized by being derived from a precursor subtilase enzymevariant having modification(s) in one or more amino acid residuessituated in positions corresponding to residues G159, S164, I165, Y167,R170, Y171 of BLS309 (in BASBPN numbering); which further has beenmodified by substitution, insertion or deletion at or in one or more ofthe residues situated in positions corresponding to residues 129, 130,131, 132, 133, 134, 135, 136 (in BASBPN numbering); whereby saidvariants exhibit increased autoproteolytic stability relative to theprecursor subtilase variant which is not modified at any of saidpositions 129-136.

[0058] U.S. Pat. No. 5,543,302 teaches that SAVINASE® has anautoproteolytic cleavage site located between residues 192-193 (inBASBPN numbering) and suggests to make modifications in a broad regionaround this site (between residue 180-210) in order to obtain a variantwith increased autoproteolytic stability.

[0059] The present inventors have confirmed this autoproteolytic sitebetween residues 192-193 (in BASBPN numbering) in both SAVINASE® andSAVINASE® variants having modification(s) in one or more amino acidresidues situated in positions corresponding to residues P129, P131,E136, G159, S164, I165, Y167, R170, Y171 of BLS309 (in BASBPNnumbering). The present inventors suggest specific modifications(mutations), in the vicinity of the autoproteolytic site betweenresidues 192-193 (in BASBPN numbering), such as S190P, and G193A (inBASBPN numbering).

[0060] Such specific mutations are not discussed or indicated in U.S.Pat. No. 5,543,302, where only a rather broad region (between residues180-210) is described and no specific mutation resulting in increasedautoproteolytic stability is disclosed in U.S. Pat. No. 5,453,302.

[0061] For further details reference is made to a working example herein(vide infra).

[0062] Accordingly, in a third aspect the invention relates to asubtilase enzyme variant characterized by being derived from a precursorsubtilase enzyme having an autoproteolytic split site between residues192 and 193 (in BASBPN numbering), which further has been modified bysubstitution, insertion or deletion at or in one or more of the residuessituated in positions corresponding to residues 189, 190, 191, 192, 193,194, 195, 196 (in BASBPN numbering); whereby said variant exhibitsincreased autoproteolytic stability relative to said precursor subtilaseenzyme.

[0063] In a fourth aspect the invention relates to a subtilase enzymevariant characterized by being derived from a precursor subtilase enzymevariant having modification(s) in one or more amino acid residuessituated in positions corresponding to residues residues P129, P131,E136, G159, S164, I165, Y167, R170, Y171 of BLS309 (in BASBPN numbering)which further has been modified by substitution, insertion or deletionat or in one or more of the residues situated in positions correspondingto residues 189, 190, 191, 192, 193, 194, 195, 196 of BLS309 (in BASBPNnumbering); whereby said variants exhibit increased autoproteolyticstability relative to the precursor subtilase variant which is notmodified at any of said positions 189-196.

[0064] In a further aspect the invention relates to DNA constructscapable of expressing the enzymes of the invention, when inserted in asuitable manner into a host cell that subsequently is brought to expressthe subtilisin enzyme(s) of the first aspect.

[0065] In a further aspect the invention relates to the production ofthe subtilisin enzymes of the invention by inserting a DNA constructaccording to the second aspect into a suitable host, cultivating thehost to express the desired subtilase enzyme, and recovering the enzymeproduct.

[0066] The invention relates, in part, but is not limited to, mutants ofthe genes expressing the subtilase sub-group I-S2 enzymes and thecorresponding enzyme variants, as indicated above.

[0067] Other subtilase gene variants encompassed by the invention aresuch as those of the subtilase subgroup I-S1, e.g. Subtilisin BPN′, andSubtilisin Carlsberg genes and ensuing variant Subtilisin BPN′,Proteinase K, and Subtilisin Carlsberg enzymes, which exhibit improvedstability in concentrated liquid detergents.

[0068] Still further subtilase gene variants encompassed by theinvention are such as Proteinase K and other genes and ensuing variantProteinase K, and other subtilase enzymes, which exhibit improvedstability in concentrated liquid detergents.

[0069] Other examples of parent subtilase enzymes that can be modifiedin accordance with the invention are listed in Table I.

[0070] Further the invention relates to the use of the mutant enzymes incleaning compositions and cleaning compositions comprising the mutantenzymes, especially detergent compositions comprising the mutantsubtilisin enzymes.

[0071] According to the invention such detergent compositions mayfurthermore comprise one or more other enzymes, such as lipases,cellulases, amylases, etc. AMINO ACIDS A = Ala = Alanine V = Val =Valine L = Leu = Leucine I = Ile = Isoleucine P = Pro = Proline F = Phe= Phenylalanine W = Trp = Tryptophan M = Met = Methionine G = Gly =Glycine S = Ser = Serine T = Thr = Threonine C = Cys = Cysteine Y = Tyr= Tyrosine N = Asn = Asparagine Q = Gln = Glutamine D = Asp = AsparticAcid E = GIu = Glutamic Acid K = Lys = Lysine R = Arg = Arginine H = His= Histidine X = Xaa = Any amino acid

[0072] NUCLEIC ACID BASES A = Adenine G = Guanine C = Cytosine T =Thymine  (only in DNA) U = Uracil  (only in RNA)

[0073] Variants

[0074] In describing the various enzyme variants produced orcontemplated according to the invention, the following nomenclatureshave been adapted for ease of reference:

[0075] Original amino acid(s) position(s) substituted amino acid(s)According to this the substitution of Glutamic acid for glycine inposition 195 is designated as:

[0076] Gly 195 Glu or G195E

[0077] a deletion of glycine in the same position is:

[0078] Gly 195 * or G195*

[0079] and insertion of an additional amino acid residue such as lysineis:

[0080] Gly 195 GlyLys or G195GK

[0081] Where a deletion in comparison with the sequence used for thenumbering is indicated, an insertion in such a position is indicated as:

[0082] *36 Asp or *36D

[0083] for insertion of an aspartic acid in position 36

[0084] Multiple mutations are separated by pluses, i.e.:

[0085] Arg 170 Tyr+Gly 195 Glu or R170Y+G195E

[0086] representing mutations in positions 170 and 195 substitutingtyrosine and glutamic acid for arginine and glycine, respectively.

[0087] Positions

[0088] In describing the variants in this application and in theappended claims use is made of the alignment of various subtilases inSiezen et al., supra. In other publications relating to subtilases otheralignments or the numbering of specific enzymes have been used. It is aroutine matter for the skilled person to establish the position of aspecific residue in the numbering used here. Reference is also made toTable I of WO 91/00345 showing an alignment of residues relevant for thepresent invention from a number of subtilases. TABLE I Presentlyestablished Subtilases (from Siezen et al., supra) acronym Organism cDNAenzyme gene PROKARYOTES Bacteria: Gram-positive Bacillus subtilis 168apr A subtilisin I168,apr ABSS168 Bacillus apr subtilisin BPN′ (NOVO)BASBPN amyloliquefaciens Bacillus subtilis DY − subtilisin DY BSSDYBacillus licheniformis + subtilisin Carlsberg BLSCAR Bacillus lentus +subtilisin 147 BLS147 Bacillus + subtilisin PB92 BAPB92 alcalophilusPB92 Bacillus sp. DSM 4828 − alkaline protease BDSM48 Bacillus YaB alealkaline elastase YaB BYSYAB Bacillus subtilis 168 epr min. extracell.prot. BSEPR Bacillus subtilis bpf bacillopeptidase F BSBPF Bacillussubtilis IFO3013 ispl intracell.ser. prot.1 BSISP1 Bacillus subtilis A50− intracell.ser. prot. BSIA50 Bacillus − extracell. ser. prot. BTFINIthuringiensis Bacillus cereus − extracell. ser. prot. BCESPRNocardiopsis dassonvillei − alkaline ser. prot. NDAPII Thermoactinomyces− thermitase TVTHER vulgaris Enterococcus cytolysin component A EFCYLAfaecallscylA Staphylococcus epiP epidermin lead. prot. SEEPIPepidermidis Streptococcus pyrogenes scpA C5a peptidase SPSCPALactococcus lactis SK11 prtP SK11 cell wall prot. LLSK11 Bacteria:Gram-neciative Dichelobacter nodosus + basic protease DNEBPR Xanthomonascampestris + extracellular prot. XCEXPR Serratia marcescens + extracell.ser. prot. SMEXSP Thermus aquaticus YT-1 pstl aqualysin I TAAQUA ThermusrT41A + T41A protease TRT41A Vibrio alginolyticus proA protease A VAPROAStreptomyces − proteinase D SRESPD rutgersensis Archaea halophilicstrain 172P1 − halophil extra. prot. ARB172 Cyanobacteria Anabaenavariabilis prcA Ca-dependent protease AVPRCA LOWER EUKARYOTES FungiTritirachium + proteinase K TAPROK album Limber Tritirachium album +proteinase R TAPROR Tritirachium album proT proteinase T TAPROTAspergillus oryzae + alkaline protease AOALPR Malbranchea pulchella −thermomycolin MPTHMY Acremonium alp alkaline protease ACALPR chrysogenumYeasts Kluyveromyces lactis kex1 Kex1 ser. proteinase KLKEX1Saccharomyces kex2 Kex2 ser. proteinase SCKEX2 cerevisiae Saccharomycesprb1 protease B SCPRB1 cerevisiae Yarrowia lipolytica xpr2alk.extracell. prot. LXPR2 HIGHER EUKARYOTES Worms Caenorhabditiselegans bli4 cuticle protease CEBLI4 Insects Drosophila (fruit fly) fur1furin 1 DMFUR1 Drosophila (fruit fly) fur2 furin 2 DMFUR2 Plants Cucumismelo (melon) − cucumisin CMCUCU Mammals Human (also rat, mouse) furfurin HSFURI Human (also mouse) + insulinoma PC2 prot. HSIPC2 Mouse +pituitary PC3 prot. MMPPC3 Human + tripeptidyl peptid.II HSTPPReferences used for Table I References to amino acid sequences(GenBank ®/EMBL Data Bank accession numbers are shown in brackets):ARB172 Kamekura and Seno, (1990) Biochem. Cell Biol. 68 352-359 (aminoacid sequencing of mature protease residues 1-35; residue 14 notdetermined). BSS168 Stahl. and Ferrari. (1984) J. Bacteriol. 158,411-418 (K01988). Yoshimoto, Qyama et al. (1488) J. Biochem. 103,1060-1065 (the mature subtilisin from B. subtilis var.amylosacchariticus differs in having T13OS and T162S). Svendsen, et al.(1986) FEBS Lell. 196,228-232 (PIR A23624; amino acid sequencing; themature alkaline mesentericopeptidase From B. mesentericus differs inhaving S85A, A88S, S89A. S183A and N259S). BASBPN Wells, et al. (1983)Nucl.Acids Res. 11 7911-7925 (X00165). Vasantha et al., (1984) J.Bacteriol. 159 811-814(K02496). BSSDY Nedkov et al. (1983)Hoppe-Seyler’s Z. Physiol. Chem. 364 1537-1540 (PIR A00969; amino acidsequencing). BLSCAR Jacobs et al. (1985) Nucleic Acids Res. 13 8913-8926(X03341). Smith et al. (1968) J. Biol. Chem. 243 2184-2191 (PIR A00968;amino acid sequencing; mature protease sequence differs in having TIO3S,P129A, S158N, N161S and S212N). BLS147 Hastrup et al. (1989) PCT PatentAppl. WO 8906279. Pub. July 13 1989. (ESPERASE ® from B. lentus). Takamiet al. (1990) Appl. Microbiol. Biotechnol., 33 519-523 (amino acidsequencing of mature alkaline protease residues 1-20 from Bacillus sp.no. AH-101; this sequence differs from BLS147 in having N11S). BABP92van der Laan et al. (1991) Appl. Environ. Microbiol. 57 901-909.(Maxacal ®). Hastrup et al. (1989) PCT Patent Appl. WO 8906279. Pub. 13Jul 1989. (subtilisin 309. SAVINASE from B. lentus differs only inhaving N87S). Godette et al. (1991) Abstracts 5th Protein SocietySymposium, June 6, Baltimore: abstract M8 (a high-alkaline protease fromB. lentus differs in having N87S, S99D, S101R, S103A, V1041 and G159S).BDSM48 Rettenmaier et al. (1990) PCT Patent Appl. WO 90/04022.Publ.April 19, 1990. BYSYAB Kaneko et al. (1989) J. Bacteriol. 1715232-5236 (M28537). BSEPR Sloma et al. (1988) J. Bacteriol. 1705557-5563 (M22407). Bruckner (1990) Mol. Gen. Genet. 221 486-490(X53307). BSBPF Sloma et al. (1990) J. Bacteriol. 172 1470-1477 (M29035;corrected). Wu et al. (1990) J. Blot. Chem. 265 6845-6850 (J05400; thissequence differs in having A169V and 586 less C-terminal residues due toa frameshift). BSISPI Koide et al. (1986) J. Bacteriol. 167 110-116(M13760). BSIA50 Strongin et al. (1978) J. Bacteriol. 133 1401-1411(amino acid sequencing of mature protease residues 1-54; residues 3. 39,40. 45, 46, 49 and 50 not determined). BTFINI Chestukhina et al. (1985)Biokhimiya 50 1724-1730 (amino acid sequencing of mature proteaseresidues 1-14 from B. thuringiensis variety israeliensis, and residuesI-16 and 223-243 from variety finitimus). Kunitate et al. (1989) Agric.Blot. Chem. 53 3251-3256 (amino acid sequencing of mature proteaseresidues 6-20 from variety kurstaki. BTKURS). BCESPR Chestukhina et al.(1985) Biokhimiya 50 1724-1730 (amino acid sequencing of mature residuesI-16 and 223-243). NDAPII Tsujibo et al. (1990) Agric. Blot. Chem. 542177-2179 (amino acid sequencing of mature residues 1-26). TVTHER Melounet al. (1985) FEBS Lett. 183 195-200 (P1RA00973; amino acid sequencingof mature protease residues 1-274). EFCYLA Segarra et al. (1991) Infect.Immun. 59 1239-1246. SEEPIP Schnell et al. (1991) personal communication(Siezen et al. (supra). SPSCPA Chen et al. (1990) J. Blot. Chem. 2653161-3167 (J05224). DNEBPR Kortt et al. (1991) Abstracts 5th ProteinSociety Symposium, June 22-26, Baltimore.abstract 576. LLSK11 Vos etal.(1989) J. Blot. Chem. 264 13579-13585 (J04962). Kok etal. (1988) Appl.Environ. Microbiol. 54 231-238 (M24767; the sequence from strain Wg2differs in 44 positions, including 18 differences in the proteasedomain, and a deletion of residues 1617-1676). Kiwaki et al. (1989)Mol.Microbiol. 3 359-369 (X14130; the sequence from strain NCD0763differs in 46 positions, including 22 in the protease domain, and adeletion of residues 1617-1 676). XCEXPR Liu et al. (1990) Mol. Gen.Genet. 220 433-440. SMEXSP Yanagida et al. (1986) J. Bacterlol. 166937-994 (M13469). TAAQUA Terada et al. (1990) J. Blot. Chem. 2656576-658 1(J054``14). TRT41A McHale et al. (1990) Abstracts 5th Eur.Congr. Biotechn. Christiansen, Munck and Viliadsen (eds), MunksgaardInt. Publishers, Copenhagen. VAPROA Deane et al. (1989) Gene 76 281-288(M25499). SRESPD Lavrenova et al. (1984) Biochemistry USSR. 49 447-454(amino acid sequencing of residues 1-23; residues 13, 18 and 19 notdetermined). AVPRCA Maldener et al (1991) Mol. Gen. Genet. 225 113-120(the published sequence has 28 uncertain residues near positions 200-210due to a frameshift reading error). TAPROK Gunkel and Gassen (1989) Eur.J. Biochem. 179 185-194 (X14688/X14689). Jany et al. (1986) J. Biol.Chem.Hoppe-Seyler 367 87(PIR A24541; amino acid sequencing; matureprotease differs in having S745G, SILST2040-208DSL and VNLL264-267FNL).TAPROR Samal et al. (1990) Mol. Microbiol. 4 1789-1792 (X56116). TAPROTSamal et al. (1989) Gene 85 329-333. AOALPR Tatsumi etal. (1989) Mol.Gen. Genet. 219 33-38. Cheevadhanarah et al. (1991) EMBL Data Library(X54726). MPTHMY Gaucher and Stevenson (1976) Methods Enzymol. 45415-433 amino acid sequencing of residues 1-28, and heapeptide LSGTSMwith active site serine). ACALPR Isogai et al. (1991) Agric. Blol. Chem.55 471-477. Stepanov et al. (1986) lnt. J. Biochem. 18 369- 375 (aminoacid sequencing of residues 1-27: the mature protease differs in havingH13[1]Q, R13[2]N and S13[6]A). KLKEX1 Tanguy-Rougeau, Wesolowski-Louveland Fukuhara (1988) FEBS left. 234 464-470 (X07038). SCKEX2 Mizuno etal. (1988) Biochem. Biophys. Res. Commun. 156 246-254(M24201). SCPRB1Moehle et al. (1987) Mol. Cell. Biol. 7 4390-4399 (M18097). YLXYPR2Davidow etal. (1987) J. Bacterlol. 169 4621-4629 (M17741). Matoba etal.(1988) Mol. Cell Biol. 8 4904-4916 (M23353). CEBL14 Peters and Rose(1991) The Worm Breeder’s Gazette 11 28. DMFUR1 Roebroek et al. (1991)FEBS Lett. 289 133-137 (X59384). DMFUR2 Roebroek et al. (1992) 26717208-17215. CMCUCU Kaneda et al. (1984) J. Biochem. 95 825-829 (aminoacid sequencing of octapeptide NIISGTSM with active site serine). HSFURIvan den Ouweland eta l. (1990) Nucl. Acids Res. 18 664 (X04329) (thesequence of mouse furin differs in 51 positions, including five in thecatalytic domain: A15E, Y21F, S223F, A232V and N258 [2]D). Misumi etal.(1990) Nucl. Acids Res. 18 6719 (X55660: the sequence of rat furindiffers in 49 positions, including three in the catalytic domain: A15E,Y21F, H24R). HSIPC2 Smeekens and Steiner (1990) J. Blot. Chem. 2652997-3000 (J05252). Seidah et al. (1990) DNA Cell Biol. 9 41 5-424 (thesequence of mouse pituitary PC2 protease differs in 23 positions,including seven in the protease domain: 14F, S42[2]Y, E45D, N76S, D133E,V134L and G239[1]D). MMPPC3 Smeekens et al. (1991) Proc. Natl. Acad. Sd.USA 88 340-344 (M58507). Seidah et al. (1990) DNA Cell Blot. 9 415-424(M55668/M55669; partial sequence). HSTPP Tomkinson and Jonsson (1991)Biochemistry 30 168-174 (J05299).

[0089] Definitions

[0090] Prior to discussing this invention in further detail, thefollowing terms will first be defined.

[0091] “Altered autolytic stability” or “altering autolytic stability”is intended to mean increasing or decreasing the autolytic stabilitycompared to that of the original protease.

[0092] “Altered autoproteolytic degradation sites” are intended toindicate altered autoproteolytic degradation sites over those of theoriginal protease. The alteration may be a formation of a completely newsite or the removal of a site in the original protease. The term“altered autoproteolytic degradation sites” is also intended to indicatealterations where an autoproteolytic site which is not a e.g. primary orsecondary cleavage site in the original protease after alteration of thedegradation pattern turns out to a e.g. a primary or a secondaryautoproteolytic site.

[0093] “Autoproteolytic cleavage site” is used to indicate a site of theprotease where the autoproteolysis of the protease is believed to takeplace. This site could e.g. be defined as being located between aminoacid residues X and Y.

[0094] Cleavage sites can be identified by preparing an aqueous solutionof a protease; rapidly inactivating the protease to prevent progressivedegradation of peptide fragments produced by autolysis; separating thepeptide fragments under conditions which prevent reactivation of theprotease; and identifying the N-terminal amino acids of the separatedfragments. For further details reference is made to a working exampleherein (vide infra).

[0095] The term “autoproteolytic cleavage site” may alternatively becalled “autoproteolytic split site”.

[0096] “Primary autoproteolytic cleavage site” is intended to mean thefirst 1-5 initial cleavage site(s) where the protease auto cleavageitself.

[0097] “Modification(s) of a subtilase variant”. The term“modification(s)” used in connection with modification(s) of a subtilasevariant as discussed herein is defined to include chemical modificationas well as genetic manipulation to reduce the rate of autolyticdegradation. The modification(s) can be by substitution, deletion and/orinsertions in or at the amino acid(s) of interest.

[0098] When the term “modification(s)” is used in connection withsubstitutions, deletion and/or insertions at or in the vicinity of anautolytic cleavage site it is defined to include modification(s) toreduce the rate of autolytic degradation. Modification should occur ator in the vicinity of the amino acids which comprise the susceptiblepolypeptide bond of those amino acids. The phrase “in the vicinity of”is defined herein to mean within three amino acids upstream ordownstream of those amino acids forming the susceptible bond.

[0099] “Random mutagenesis”. is intended to be understood in aconventional manner, i.e. to indicate an introduction of one or moremutations at random positions of the parent enzyme or introduction ofrandom amino acid residues in selected positions or regions of theparent enzyme. The random mutagenesis is normally accompanied by ascreening which allows to select for mutated enzymes which, as comparedwith the parent enzyme, has improved properties. Suitable techniques forintroducing random mutations and screening for improved properties arediscussed in further detail herein.

[0100] “Wash performance” means the ability of an enzyme to catalyze thedegradation of various naturally occurring substrates present on theobjects to be cleaned during e.g. wash is often referred to as itswashing ability, washability, detergency, or wash performance.Throughout this application the term wash performance will be used toencompass this property.

[0101] “SAVINASE®” is marketed by NOVO NORDISK A/S. It is subtilisin 309from B. lentus and differs from BABP92 only in having N87S (see Table Iherein).

[0102] “Precursor subtilase” is a subtilase defined according to Siezenet al. (Protein Engineering 4:719-737 (1991)). For further details seesection named “SUBTILASES” described herein. A precursor subtilase mayalso be a subtilase isolated from a natural source, wherein subsequentmodification have been made while retaining the characteristic of asubtilase.

[0103] “Substrate” used in connection with a substrate for a proteaseshould be interpreted in its broadest form as comprising a compoundcontaining at least one peptide bond susceptible to hydrolysis by asubtilisin protease.

[0104] “Product” used in connection with a product derived from aprotease enzymatic reaction should in the context of this invention beinterpreted to include the products of a hydrolysis reaction involving asubtilisin protease. A product may be the substrate in a subsequenthydrolysis reaction.

[0105] “Subtilase variant” or “mutated subtilase” means a subtilase thathas been produced by an organism which is expressing a mutant genederived from a parent microorganism which possessed an original orparent gene and which produced a corresponding parent enzyme, the parentgene having been mutated in order to produce the mutant gene from whichsaid mutated subtilisin protease is produced when expressed in asuitable host.

DETAILED DESCRIPTION OF THE INVENTION

[0106] Subtilase variants which have an altered autoproteolyticdegradation pattern:

[0107] According to the invention any precursor subtilase having aprimary autoproteolytic site located at or close to residues 132-133 (inBASBPN numbering), will exhibit increased autolytic stability by beingmodified by substitutions at or in the vicinity (between residue129-136) of said autolytic cleavage site located between residues132-133 (in BASBPN numbering).

[0108] According to the invention a precursor subtilase having such aprimary autolytic cleavage site located between residues 132-133 (inBASBPN numbering), can be a subtilase variant or a wildtype subtilase.The wildtype subtilase may be any of those indicated in Table I havingthe above specified cleavage site. The subtilase variant may be but isnot limited to the variant discussed in Table II. It is presentlybelieved that other subtilase variants than those discussed in Table IImay also have a primary autolytic cleavage site located between residues132 and 133 (in BASBPN numbering).

[0109] According to the invention a subtilase variant havingmodification(s) in one or more of the amino residues mentioned below(See Table II) results in altered autoproteolytic degradation sites,preferably a new primary autoproteolytic site located at or close toresidues 132-133 (in BASBPN numbering).

[0110] According to the invention a subtilase variant having one or moremodification(s) in any of the amino acid residues shown in Table II willexhibit increased autolytic stability by being modified by substitutionsat or in the vicinity (between residue 129-136) of said autolyticcleavage site located between residues 132-133 (in BASBPN numbering).

[0111] A subtilase variant having one or more modification(s) in any ofthe amino acid residues shown in Table II, will exhibit increasedautolytic stability too by being modified by substitutions at or in thevicinity (between residue 190-196) of the autolytic cleavage sitelocated between residues 192-193 (in BASBPN numbering) and according tothe invention a mutual benefit can be obtained by combiningmodifications in the vicinity around of both the autoproteolyticdegradation sites between 132-133 and 192-193. TABLE II Residues whichwhen modified give rise to a subtilase variant with an alteredautoproteolytic degradation pattern: Pos\Enz. BASBPN BLSCAR BLS309BLS147 TVTHER 129 P P P T T 131 G G P G G 136 K K E E Q 159 S S G Q T164 T T S G A 165 V I I V P 167 Y Y Y Y Y 170 K K R R Y 171 Y Y Y Y Y

[0112] Further Table III illustrates the specific amino acid residues inthe vicinity of the mentioned autolytic cleavage site located betweenresidues 132-133 (In BASBPN numbering). It is obvious that such asimilar or larger tables covering other subtilases may easily beproduced by the skilled person. TABLE III Residues in the vicinity ofthe autoproteolytic site located between residue 132-133: (In BASBPNnumbering). Pos\Enz. BASBPN BLSCAR BLS309 BLS147 TVTHER 129 P P P T T130 S S S S V 131 G G P G G 132 S S S S N 133 A T A S S 134 A A T T G135 L M L L L 136 K K E E Q

[0113] A similar table illustrating residues in the vicinity of theautoproteolytic site located between residue 192-193 could easily bemade by a person skilled in the art.

[0114] Consequently the invention relates to subtilase variants havingmodifications in one or more of the amino residues illustrated in TableII in which the amino acid sequence further has been modified at one ormore of the amino acid residue in the vicinity of the autoproteolyticsite located between positions 132-133 (in BASBPN numbering) (i.e.positions 129, 130, 131, 132, 133, 134, 135, 136); or

[0115] Subtilase variants having modifications in one or more of theamino residues illustrated in Table II which further been modified inthe vicinity of the autoproteolytic site located between positions192-193 (in BASBPN numbering) (i.e. positions 190, 191, 192, 193, 194,195, 196); or

[0116] Subtilase variants having modifications in one or more of theamino residues illustrated in Table II which further have been modifiedin the vicinity of both of the two autoproteolytic sites mentionedimmediately above.

[0117] According to the invention a number of specific modifications inthe vicinity of both the autoproteolytic sites located between 132-133and 192-193 (in BASBPN numbering) or each of the sites will provideincreased autoproteolytic stability of the subtilase.

[0118] In principle the modification may be a replacement of an aminoacid residue located in the vicinity of the cleavage site with any ofthe other 19 possible amino acid residues resulting in an increasedautoproteolytic stability of the resulting variant. Similarly, themodification may be an insertion of one or more of any of the 20possible amino acid residues in the vicinity of the cleavage siteresulting in an increased autoproteolytic stability of the resultingvariant. Further the modification may be a deletion of any of the aminoacid residue located in the vicinity of the cleavage site resulting inan increased autoproteolytic stability of the resulting variant.

[0119] A strategy to identify the specific modifications giving rise toincreased autoproteolytic stability is to make localized randommutagenesis in the whole region in the vicinity of one of the siteand/or both of the sites (e.g. localized random mutagenesis in allresidues between 129-136 and/or 189-196) followed by a screening assayto identify the specific modifications giving rise to increasedstability. For illustration of this strategy reference is made toworking examples herein (vide infra).

[0120] A number of specific mutations, giving rise to increasedautoproteolytic stability, are indicated herein (See section “B” and “C”below).

[0121] By e.g. looking at Table II or III and applying the principle ofthe invention a number of candidates for subtilase variants withincreased autoproteolytic stability becomes clear.

[0122] For both precursor subtilase variants of BASBPN and BLSCAR havingan autoproteolytic split site between residues 132-133 (in BASBPNnumbering) it is appropriate to make substitutions in any of thepositions in the vicinity of this autoproteolytic site in order to makevariants with increased autoproteolytic stability.

[0123] In the context of this invention a subtilase is defined inaccordance with Siezen et al. supra. In a more narrow sense, applicableto many embodiments of the invention, the subtilases of interest arethose belonging to the subgroups I-S1 and I-S2. In a more specificsense, many of the embodiments of the invention relate to serineproteases of gram-positive bacteria which can be brought intosubstantially unambiguous homology in their primary structure, with thesubtilases listed in Table I above.

[0124] The present invention also comprises any one or moresubstitutions in the above mentioned positions in combination with anyother substitution, deletion or addition to the amino acid sequence ofthe parent enzyme. Especially combinations with other substitutionsknown to provide improved properties to the enzyme are envisaged.

[0125] Such combinations comprise the positions: 222 (improve oxidationstability), 218 (improves thermal stability), substitutions in theCa-binding sites stabilising the enzyme, e.g. position 76, and manyother apparent from the prior art.

[0126] Furthermore combinations with the variants mentioned in EP 405901 are also contemplated specifically.

[0127] Variants

[0128] A: Single Variants with an Altered Autoproteolytic Cleavage SiteBetween 132-133: (in BASBPN Numbering).

[0129] The single variants comprise one or more of the mutationsmentioned below: Subtilisin BPN′, Subtilisin Carlsberg, Subtilisin 168,and Subtilisin DY variants:

[0130] A129V, A129I, A129L, A129M, A129F,

[0131] G131V, G131I, G131L, G131M, G131F,

[0132] K136V, K136I, K136L, K136M, K136F,

[0133] S159V, S159I, S159L, S159M, S159F,

[0134] T164V, T164I, T164L, T164M, T164F,

[0135] Y167V, Y167I, Y167L, Y167M, Y167F,

[0136] K170V, K170I, K170L, K170M, K170F,

[0137] Y171V, Y171I, Y171L, Y171M, Y171F.

[0138] Thermitase Variants:

[0139] A129V, A129I, A129L, A129M, A129F,

[0140] G131V, G131I, G131L, G131M, G131F,

[0141] Q136V, Q136I, Q136L, Q136M, Q136F,

[0142] T159V, T159I, T159L, T159M, T159F,

[0143] A164V, A164I, A164L, A164M, A164F,

[0144] Y167V, Y167I, Y167L, Y167M, Y167F,

[0145] Y171V, Y171I, Y171L, Y171M, Y171 F,

[0146] Y170V, Y170I, Y170L, Y170M, Y170F.

[0147] Subtilisin 309, Subtilisin 147, and Bacillus PB92 ProteaseVariants:

[0148] T129V, T129I, T129L, T129M, T129F,

[0149] G131V, G131I, G131L, G131M, G131F,

[0150] E136V, E136I, E136L, E136M, E136F,

[0151] G159V, G159I, G159L, G159M, G159F,

[0152] G164V, G164I, G164L, G164M, G164F, (BLS147)

[0153] S164V, S164I, S164L, S164M, S164F, (BLS309 AND BAPB92)

[0154] Y167A, Y167H, Y167N, Y167P, Y167C, Y167W, Y167Q, Y167S, Y167T,Y167G,

[0155] Y167V, Y167I, Y167L, Y167M, Y167F

[0156] R170W, R170A, R170H, R170N, R170P, R170Q, R170S, R170T, R170Y(disclaimed for BLS309), R170V (disclaimed for BAPB92), R170I(disclaimed for BAPB92), R170L, R170M (disclaimed for BAPB92), R170F,R170G, R170C,

[0157] Y171A, Y171H, Y171N, Y171P, Y171C, Y171W, Y171Q, Y171S, Y171T,Y171G,

[0158] Y171V, Y171I, Y171L, Y171M, Y171F.

[0159] B: Variants Modified in the Vicinity of the AutoproteolyticCleavage Site Located between Positions 132-133.

[0160] Any of the single variant mentioned under section “A:” abovewhich further comprise one or more of any of the following mutations:

[0161] P129D, P129E, P129A (BLS309)

[0162] S130D, S130E, S130A (BLS309)

[0163] P131G, P131D, P131A (BLS309)

[0164] S132C, S132A, S132P (BLS309)

[0165] A133P (BLS309)

[0166] T134C, T134P, T134A (BLS309)

[0167] L135P, L135A (BLS309)

[0168] E136A, E136P, E136K (BLS309)

[0169] V104C+S132C (BLS309)

[0170] V104C+T134C (BLS309)

[0171] A108C+T134C (BLS309)

[0172] C: Variants Modified in the Vicinity of the AutoproteolyticCleavage Site Located between Positions 192-193.

[0173] Any of the single variant mentioned under section “A:” abovewhich further comprise one or more of any of the following mutations:

[0174] F189A, F189G, F189D, F189R, F189Y, F189E, F189N (BLS309)

[0175] S190P, S190D, S190T (BLS309)

[0176] Q191S, Q191T, Q191N, Q191A, 0191L, Q191D, Q191W (BLS309)

[0177] Y192A, Y192P, Y192D, Y192E, Y192V (BLS309)

[0178] G193A, G193N, G193P (BLS309)

[0179] A194P, A194D (BLS309) G195E (BLS309)

[0180] L196A (BLS309)

[0181] D: Variants Modified in the Vicinity of both the AutoproteolyticCleavage Sites Located between Positions 132-133 and 192-193.

[0182] Any of the single variant mentioned under section “A:” abovewhich further comprise a combination of one or more of any of themutations mentioned under section “B” and/or one or more of any of themutations mentioned under section “C” above.

[0183] E: Further Combination Variants:

[0184] Any of the above variants mentioned under section “B”, “C”,and/or “D” are contemplated to prove advantageous if combined with othermutations in any of the positions:

[0185] 27, 36, 57, 76, 97, 101, 104, 120, 123, 206, 218, 222, 224, 235and 274.

[0186] Specifically the mutations in the following BLS309 and BAPB92variants are considered appropriate for combination:

[0187] K27R, *36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D,N123S, A194P, Q206E, N218S, M222S, M222A, T224S, K235L and T274A.

[0188] Also such variants comprising any one or two of the substitutionsX167V, X167M, X167F, X167L, X167I, X170V, X170M, X170F, X170L, and/orX170I in combination with any one or more of the other substitutions,deletions and/or insertions mentioned above are considered advantageousto combine with any of the mutations mentioned under section “A”, “B”and/or “C”.

[0189] Furthermore variants comprising any of the mutations V104N+S101G,K27R+V104Y+N123S+T274A, or N76D+V104A or other combinations of thesemutations (V104N, S101 G, K27R, V104Y, N123S, T274A, N76D, V104A), incombination with any one or more of the substitutions, deletions and/orinsertions mentioned above under section “A”, “B”, and/or “C” are deemedto exhibit improved properties.

[0190] Specific combinations to be mentioned are: A: Y167I + R170L +A133P B: Y167I + R170L + T134P C: Y167I + R170L + A133P + T134P D:Y167I + R170L + V140C + S132C E: Y167I + R170L + A108C + T134C F:Y167A + R170S + F189A G: Y167A + R170S + Y192A H: Y167A + R170S + Y192PI: Y167A + R170S + Y192A + A194P J: Y167A + R170S + Y192P + A194P K:Y167A + R170S + F189G L: Y167A + R170S + F189E M: Y167A + R170S + F189RN: Y1671 + R170L M: Y1671 + R170L + A194P 0: Y167A + R170S + A194P P:Y167A + R170L + A194P Q: Y167A + R170N + A194P R: V104C + S132C +Y1671 + R170L S: A108C + T134C + Y1671 + R170L T: V104C + S132C +Y167A + R170S U: V104C + S132C + Y167A + R170L V: V104C + S132C +Y167A + R170N X: A133D + Y167I + R170L Y: P129K + Y1671 + R170L Z:A133P + Y167A + R170S + A194P AA: T134P + Y167A + R170S + A194P BB:A133P + T134P + Y167A + R170S + A194P CC: A133P + Y167A + R170N + A194PDD: T134P + Y167A + R170N + A194P EE: A133P + T134P + Y167A + R170N +A194P FF: A133P + Y167A + R170L GG: P129K + P131H + Y167I + R170L HH:A133P + Y167A + R170S II: A133P + Y167A + R170N JJ: Y167A + R170S +F189K KK: V104C + T134C + Y167A + R170S

[0191] Method for Producing Mutations in Subtilase Genes

[0192] Many methods for introducing mutations into genes are well knownin the art. After a brief discussion of cloning subtilase genes, methodsfor generating mutations in both random sites, and specific sites,within the subtilase gene will be discussed.

[0193] Cloning a Subtilase Gene

[0194] The gene encoding a subtilase may be cloned from any of theorganisms indicated in Table I, especially gram-positive bacteria orfungus, by various methods, well known in the art. First a genomic,and/or cDNA library of DNA must be constructed using chromosomal DNA ormessenger RNA from the organism that produces the subtilase to bestudied. Then, if the amino-acid sequence of the subtilase is known,homologous, labelled oligonucleotide probes may be synthesized and usedto identify subtilisin-encoding clones from a genomic library ofbacterial DNA, or from a cDNA library. Alternatively, a labelledoligonucleotide probe containing sequences homologous to subtilase fromanother strain of bacteria or organism could be used as a probe toidentify subtilase-encoding clones, using hybridization and washingconditions of lower stringency.

[0195] Yet another method for identifying subtilase-producing cloneswould involve inserting fragments of genomic DNA into an expressionvector, such as a plasmid, transforming protease-negative bacteria withthe resulting genomic DNA library, and then plating the transformedbacteria onto agar containing a substrate for subtilase, such as skimmilk. Those bacteria containing subtilase-bearing plasmid will producecolonies surrounded by a halo of clear agar, due to digestion of theskim milk by excreted subtilase.

[0196] Generation of Random Mutations in the Subtilase Gene

[0197] Once the subtilase gene has been cloned into a suitable vector,such as a plasmid, several methods can be used to introduce randommutations into the gene.

[0198] For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents.

[0199] The mutagenizing agent may, e.g., be one which inducestransitions, transversions, inversions, scrambling, deletions, and/orinsertions.

[0200] Examples of a physical or chemical mutagenizing agent suitablefor the present purpose includes ultraviolet (UV) irradiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodiumbisulphite, formic acid, and nucleotide analogues.

[0201] When such agents are used the mutagenesis is typically performedby incubating the DNA sequence encoding the parent enzyme to bemutagenized in the presence of the mutagenizing agent of choice undersuitable conditions for the mutagenesis to take place, and selecting formutated DNA having the desired properties.

[0202] When the mutagenesis is performed by the use of anoligonucleotide, the oligonucleotide may be doped or spiked with thethree non-parent nucleotides during the synthesis of the oligonucleotideat the positions wanted to be changed. The doping or spiking may be doneso that codons for unwanted amino acids are avoided. The doped or spikedoligonucleotide can be incorporated into the DNA encoding the proteaseenzyme by any published technique using e.g. PCR, LCR or any DNApolymerase and ligase.

[0203] When PCR generated mutagenesis is used either a chemicallytreated or non-treated gene encoding a parent protease enzyme issubjected to PCR under conditions that increases the misincorporation ofnucleotides (Deshler 1992, Leung et al. 1989).

[0204] A mutator strain of E. coli (Fowler et al. 1974), S. cereviciaeor any other microbial organism may be used for the random mutagenesisof the DNA encoding the protease enzyme by e.g. transforming a plasmidcontaining the parent enzyme into the mutator strain, growing themutator strain with the plasmid and isolating the mutated plasmid fromthe mutator strain. The mutated plasmid may subsequently be transformedinto the expression organism.

[0205] The DNA sequence to be mutagenized may conveniently be present ina genomic or cDNA library prepared from an organism expressing theparent protease enzyme. Alternatively, the DNA sequence may be presenton a suitable vector such as a plasmid or a bacteriophage, which as suchmay be incubated with or otherwise exposed to the mutagenizing agent.The DNA to be mutagenized may also be present in a host cell either bybeing integrated in the genome of said cell or by being present on avector harboured in the cell. Finally, the DNA to be mutagenized may bein isolated form. The DNA sequence to be subjected to random mutagenesisis preferably a cDNA or a genomic DNA sequence.

[0206] The random mutagenesis may advantageously be located to a part ofthe parent protease in question. This may, e.g., be advantageous when acertain region of the enzyme has been identified to be of particularimportance for a given property of the enzyme, and which, when modified,is expected to result in a variant having improved properties. Suchregion may normally be identified when the tertiary structure of theparent enzyme has been elucidated and related to the function of theenzyme.

[0207] The localized random mutagenesis is conveniently performed by useof PCR generated mutagenesis techniques as described above or any othersuitable technique known in the art.

[0208] Alternatively, the DNA sequence encoding the part of the DNAsequence to be modified may be isolated, e.g. by being inserted into asuitable vector, and said part may subsequently be subjected tomutagenesis by use of any of the mutagenesis methods discussed above.

[0209] The localized random mutagenesis may be performed in one or moreof these regions, and is preferably performed in at least two of theregions.

[0210] Generation of Site Directed Mutations in the Subtilase Gene

[0211] Once the subtilase gene has been cloned, and desirable sites formutation identified and the residue to substitute for the original oneshave been decided, these mutations can be introduced using syntheticoligonucleotides. These oligonucleotides contain nucleotide sequencesflanking the desired mutation sites; mutant nucleotides are insertedduring oligonucleotide synthesis. In a preferred method, Site-directedmutagenesis is done by the “Unique site elimination (USE)” or the“Uracil-USE” technique described respectively by Deng et al. (Anal.Biochem. 200:81-88 (1992)) and Markvardsen et al. (BioTechniques18(3):371-372 (1995)).

[0212] Recombinant Expression Vectors

[0213] A recombinant vector comprising a DNA construct encoding theenzyme of the invention may be any vector which may conveniently besubjected to recombinant DNA procedures, and the choice of vector willoften depend on the host cell into which it is to be introduced. Thus,the vector may be an autonomously replicating vector, i.e. a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g. a plasmid. Alternatively,the vector may be one which, when introduced into a host cell, isintegrated into the host cell genome in part or in its entirety andreplicated together with the chromosome(s) into which it has beenintegrated.

[0214] The vector is preferably an expression vector in which the DNAsequence encoding the enzyme of the invention is operably linked toadditional segments required for transcription of the DNA. In general,the expression vector is derived from plasmid or viral DNA, or maycontain elements of both. The term, “operably linked” indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in a promoter andproceeds through the DNA sequence coding for the enzyme.

[0215] The promoter may be any DNA sequence which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.

[0216] Examples of suitable promoters for use in bacterial host cellsinclude the promoter of the Bacillus stearothermophilus maltogenicamylase gene, the Bacillus licheniformis alpha-amylase gene, theBacillus amyloliquefaciens alpha-amylase gene, the Bacillus subtilisalkaline protease gen, or the Bacillus pumilus xylosidase gene, or thephage Lambda PR or PL promoters or the E. coli lac, trp or tacpromoters.

[0217] The DNA sequence encoding the enzyme of the invention may also,if necessary, be operably connected to a suitable terminator.

[0218] The recombinant vector of the invention may further comprise aDNA sequence enabling the vector to replicate in the host cell inquestion.

[0219] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, or a geneencoding resistance to e.g. antibiotics like kanamycin, chloramphenicol,erythromycin, tetracycline, spectinomycine, or the like, or resistanceto heavy metals or herbicides.

[0220] To direct an enzyme of the present invention into the secretorypathway of the host cells, a secretory signal sequence (also known as aleader sequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the enzyme in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe enzyme. The secretory signal sequence may be that normallyassociated with the enzyme or may be from a gene encoding anothersecreted protein.

[0221] The procedures used to ligate the DNA sequences coding for thepresent enzyme, the promoter and optionally the terminator and/orsecretory signal sequence, respectively, or to assemble these sequencesby suitable PCR amplification schemes, and to insert them into suitablevectors containing the information necessary for replication orintegration, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., op. cit.).

[0222] Host Cell

[0223] The DNA sequence encoding the present enzyme introduced into thehost cell may be either homologous or heterologous to the host inquestion. If homologous to the host cell, i.e. produced by the host cellin nature, it will typically be operably connected to another promotersequence or, if applicable, another secretory signal sequence and/orterminator sequence than in its natural environment. The term“homologous” is intended to include a DNA sequence encoding an enzymenative to the host organism in question. The term “heterologous” isintended to include a DNA sequence not expressed by the host cell innature. Thus, the DNA sequence may be from another organism, or it maybe a synthetic sequence.

[0224] The host cell into which the DNA construct or the recombinantvector of the invention is introduced may be any cell which is capableof producing the present enzyme and includes bacteria, yeast, fungi andhigher eukaryotic cells.

[0225] Examples of bacterial host cells which, on cultivation, arecapable of producing the enzyme of the invention are gram-positivebacteria such as strains of Bacillus, such as strains of B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus, B. megatherium or B. thuringiensis, or strains of Streptomyces,such as S. lividans or S. murinus, or gram-negative bacteria such asEcherichia coli. The transformation of the bacteria may be effected byprotoplast transformation, electroporation, conjugation, or by usingcompetent cells in a manner known per se (cf. Sambrook et al., supra).

[0226] When expressing the enzyme in bacteria such as E. coli, theenzyme may be retained in the cytoplasm, typically as insoluble granules(known as inclusion bodies), or may be directed to the periplasmic spaceby a bacterial secretion sequence. In the former case, the cells arelysed and the granules are recovered and denatured after which theenzyme is refolded by diluting the denaturing agent. In the latter case,the enzyme may be recovered from the periplasmic space by disrupting thecells, e.g. by sonication or osmotic shock, to release the contents ofthe periplasmic space and recovering the enzyme.

[0227] When expressing the enzyme in gram-positive bacteria such asBacillus or Streptomyces strains, the enzyme may be retained in thecytoplasm, or may be directed to the extracellular medium by a bacterialsecretion sequence. In the latter case, the enzyme may be recovered fromthe medium as described below.

[0228] Method of Producing Subtilase

[0229] The present invention provides a method of producing an isolatedenzyme according to the invention, wherein a suitable host cell, whichhas been transformed with a DNA sequence encoding the enzyme, iscultured under conditions permitting the production of the enzyme, andthe resulting enzyme is recovered from the culture.

[0230] As defined herein, an isolated polypeptide (e.g. an enzyme) is apolypeptide which is essentially free of other non-subtilasepolypeptides, e.g., at least about 20% pure, preferably at least about40% pure, more preferably about 60% pure, even more preferably about 80%pure, most preferably about 90% pure, and even most preferably about 95%pure, as determined by SDS-PAGE.

[0231] The term “isolated polypeptide” may alternatively be termed“purified polypeptide”.

[0232] When an expression vector comprising a DNA sequence encoding theenzyme is transformed into a heterologous host cell it is possible toenable heterologous recombinant production of the enzyme of theinvention.

[0233] Thereby it is possible to make a highly purified subtilasecomposition, characterized in being free from homologous impurities.

[0234] In this context homologous impurities means any impurities (e.g.other polypeptides than the enzyme of the invention) which originatefrom the homologous cell where the enzyme of the invention is originallyobtained from.

[0235] The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed subtilase may conveniently be secreted into the culture mediumand may be recovered therefrom by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulphate, followed by chromatographic procedures suchas ion exchange chromatography, affinity chromatography, or the like.

[0236] Detergent Compositions Comprising the Mutant Enzymes

[0237] The present invention also comprises the use of the mutantenzymes of the invention in cleaning and detergent compositions and suchcompositions comprising the mutant subtilisin enzymes. Such cleaning anddetergent compositions are described in further details below.

[0238] Detergent Disclosure and Examples

[0239] Surfactant System

[0240] The detergent compositions according to the present inventioncomprise a surfactant system, wherein the surfactant can be selectedfrom nonionic and/or anionic and/or cationic and/or ampholytic and/orzwitterionic and/or semi-polar surfactants.

[0241] The surfactant is typically present at a level from 0.1% to 60%by weight.

[0242] The surfactant is preferably formulated to be compatible withenzyme components present in the composition. In liquid or gelcompositions the surfactant is most preferably formulated in such a waythat it promotes, or at least does not degrade, the stability of anyenzyme in these compositions.

[0243] Preferred systems to be used according to the present inven-tioncomprise as a surfactant one or more of the nonionic and/or anionicsurfactants described herein.

[0244] Polyethylene, polypropylene, and polybutylene oxide conden-satesof alkyl phenols are suitable for use as the nonionic surfactant of thesurfactant systems of the present invention, with the polyethylene oxidecondensates being preferred. These compounds include the condensationproducts of alkyl phenols having an alkyl group containing from about 6to about 14 carbon atoms, preferably from about 8 to about 14 carbonatoms, in either a straight chain or branched-chain configuration withthe alkylene oxide. In a preferred embodiment, the ethylene oxide ispresent in an amount equal to from about 2 to about 25 moles, morepreferably from about 3 to about 15 moles, of ethylene oxide per mole ofalkyl phenol. Commercially available nonionic surfactants of this typeinclude Igepal™ CO-630, marketed by the GAF Corporation; and Triton™X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company.These surfactants are commonly referred to as alkylphenol alkoxylates(e.g., alkyl phenol ethoxylates).

[0245] The condensation products of primary and secondary aliphaticalcohols with about 1 to about 25 moles of ethylene oxide are suitablefor use as the nonionic surfactant of the nonionic surfactant systems ofthe present invention. The alkyl chain of the aliphatic alcohol caneither be straight or branched, primary or secondary, and generallycontains from about 8 to about 22 carbon atoms. Preferred are thecondensation products of alcohols having an alkyl group containing fromabout 8 to about 20 carbon atoms, more preferably from about 10 to about18 carbon atoms, with from about 2 to about 10 moles of ethylene oxideper mole of alcohol. About 2 to about 7 moles of ethylene oxide and mostpreferably from 2 to 5 moles of ethylene oxide per mole of alcohol arepresent in said condensation products. Examples of commerciallyavailable nonionic surfactants of this type include Tergitol™ 15 S-9(The condensation product of C₁₁-C₁₅ linear alcohol with 9 molesethylene oxide), Tergitol™ 24-L-6 NMW (the condensation product ofC₁₂-C₁₄ primary alcohol with 6 moles ethylene oxide with a narrowmolecular weight distribution), both marketed by Union CarbideCorporation; Neodol™ 45-9 (the condensation product of C₁₄-C₁₅ linearalcohol with 9 moles of ethylene oxide), Neodol™ 23-3 (the condensationproduct of C₁₂-C₁₃ linear alcohol with 3.0 moles of ethylene oxide),Neodol™ 45-7 (the condensation product of C₁₄-C₁₅ linear alcohol with 7moles of ethylene oxide), Neodol™ 45-5 (the condensation product ofC₁₄-C₁₅ linear alcohol with 5 moles of ethylene oxide) marketed by ShellChemical Company, Kyro™ EOB (the condensation product of C₁₃-C₁₅ alcoholwith 9 moles ethylene oxide), marketed by The Procter & Gamble Company,and Genapol LA 050 (the condensation product of C₁₂-C₁₄ alcohol with 5moles of ethylene oxide) marketed by Hoechst. Preferred range of HLB inthese products is from 8-11 and most preferred from 8-10.

[0246] Also useful as the nonionic surfactant of the surfactant systemsof the present invention are alkylpolysaccharides disclosed in U.S. Pat.No. 4,565,647, having a hydrophobic group containing from about 6 toabout 30 carbon atoms, preferably from about 10 to about 16 carbon atomsand a polysaccharide, e.g. a polyglycoside, hydrophilic group containingfrom about 1.3 to about 10, preferably from about 1.3 to about 3, mostpreferably from about 1.3 to about 2.7 saccharide units. Any reducingsaccharide containing 5 or 6 carbon atoms can be used, e.g., glucose,galactose and galactosyl moieties can be substituted for the glucosylmoieties (optionally the hydrophobic group is attached at the 2-, 3-,4-, etc. positions thus giving a glucose or galactose as opposed to aglucoside or galactoside). The intersaccharide bonds can be, e.g.,between the one position of the additional saccharide units and the 2-,3-, 4-, and/or 6-positions on the preceding saccharide units.

[0247] The preferred alkylpolyglycosides have the formula

R²O(C_(n)H_(2n)O)_(t)(glycosyl)_(x)

[0248] wherein R² is selected from the group consisting of alkyl,alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof inwhich the alkyl groups contain from about 10 to about 18, preferablyfrom about 12 to about 14, carbon atoms; n is 2 or 3, preferably 2; t isfrom 0 to about 10, preferably 0; and x is from about 1.3 to about 10,preferably from about 1.3 to about 3, most preferably from about 1.3 toabout 2.7. The glycosyl is preferably derived from glucose. To preparethese compounds, the alcohol or alkylpolyethoxy alcohol is formed firstand then reacted with glucose, or a source of glucose, to form theglucoside (attachment at the 1-position). The additional glycosyl unitscan then be attached between their 1-position and the preceding glycosylunits 2-, 3-, 4-, and/or 6-position, preferably predominantly the2-position.

[0249] The condensation products of ethylene oxide with a hydrophobicbase formed by the condensation of propylene oxide with propylene glycolare also suitable for use as the additional nonionic surfactant systemsof the present invention. The hydrophobic portion of these compoundswill preferably have a molecular weight from about 1500 to about 1800and will exhibit water insolubility. The addition of polyoxyethylenemoieties to this hydrophobic portion tends to increase the watersolubility of the molecule as a whole, and the liquid character of theproduct is retained up to the point where the polyoxyethylene content isabout 50% of the total weight of the condensation product, whichcorresponds to condensation with up to about 40 moles of ethylene oxide.Examples of compounds of this type include certain of the commerciallyavailable Pluronic™ surfactants, marketed by BASF.

[0250] Also suitable for use as the nonionic surfactant of the nonionicsurfactant system of the present invention, are the condensationproducts of ethylene oxide with the product resulting from the reactionof propylene oxide and ethylenediamine. The hydrophobic moiety of theseproducts consists of the reaction product of ethylenediamine and excesspropylene oxide, and generally has a molecular weight of from about 2500to about 3000. This hydrophobic moiety is condensed with ethylene oxideto the extent that the condensation product contains from about 40% toabout 80% by weight of polyoxyethylene and has a molecular weight offrom about 5,000 to about 11,000. Examples of this type of nonionicsurfactant include certain of the commercially available Tetronic™compounds, marketed by BASF.

[0251] Preferred for use as the nonionic surfactant of the surfactantsystems of the present invention are polyethylene oxide condensates ofalkyl phenols, condensation products of primary and secondary aliphaticalcohols with from about 1 to about 25 moles of ethyleneoxide,alkylpolysaccharides, and mixtures hereof. Most preferred are C₈-C₁₄alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C₈-C₁₈alcohol ethoxylates (preferably C₁₀ avg.) having from 2 to 10 ethoxygroups, and mixtures thereof.

[0252] Highly preferred nonionic surfactants are polyhydroxy fatty acidamide surfactants of the formula

[0253] wherein R¹ is H, or R¹ is C₁₋₄ hydrocarbyl, 2-hydroxyethyl,2-hydroxypropyl or a mixture thereof, R² is C₅₋₃₁ hydrocarbyl, and Z isa polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least3 hydroxyls directly connected to the chain, or an alkoxylatedderivative thereof. Preferably, R¹ is methyl, R² is straight C₁₁₋₁₅alkyl or C₁₆₋₁₈ alkyl or alkenyl chain such as coconut alkyl or mixturesthereof, and Z is derived from a reducing sugar such as glucose,fructose, maltose or lactose, in a reductive amination reaction.

[0254] Highly preferred anionic surfactants include alkyl alkoxylatedsulfate surfactants. Examples hereof are water soluble salts or acids ofthe formula RO(A)_(m)SO3M wherein R is an unsubstituted C₁₀-C₂₄ alkyl orhydroxyalkyl group having a C₁₀-C₂₄ alkyl component, preferably aC₁₂-C₂₀ alkyl or hydro-xyalkyl, more preferably C₁₂-C₁₈ alkyl orhydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero,typically between about 0.5 and about 6, more preferably between about0.5 and about 3, and M is H or a cation which can be, for example, ametal cation (e.g., sodium, potassium, lithium, calcium, magnesium,etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylatedsulfates as well as alkyl propoxylated sulfates are contemplated herein.Specific examples of substituted ammonium cations include methyl-,dimethyl, trimethyl-ammonium cations and quaternary ammonium cationssuch as tetramethyl-ammonium and dimethyl piperdinium cations and thosederived from alkylamines such as ethylamine, diethylamine,triethylamine, mixtures thereof, and the like. Exemplary surfactants areC₁₂-C₁₈ alkyl polyethoxylate (1.0) sulfate (C₁₂-C₁₈E(1.0)M), C₁₂-C₁₈alkyl polyethoxylate (2.25) sulfate (C₁₂-C₁₈(2.25)M, and C₁₂-C₁₈ alkylpolyethoxylate (3.0) sulfate (C₁₂-C₁₈E(3.0)M), and C₁₂-C₁₈ alkylpolyethoxylate (4.0) sulfate (C₁₂-C₁₈E(4.0)M), wherein M is convenientlyselected from sodium and potassium.

[0255] Suitable anionic surfactants to be used are alkyl ester sulfonatesurfactants including linear esters of C₈-C₂₀ carboxylic acids (i.e.,fatty acids) which are sulfonated with gaseous SO₃ according to “TheJournal of the American Oil Chemists Society”, 52 (1975), pp. 323-329.Suitable starting materials would include natural fatty substances asderived from tallow, palm oil, etc.

[0256] The preferred alkyl ester sulfonate surfactant, especially forlaundry applications, comprise alkyl ester sulfonate surfactants of thestructural formula:

[0257] wherein R³ is a C₈-C₂₀ hydrocarbyl, preferably an alkyl, orcombination thereof, R⁴ is a C₁-C₆ hydrocarbyl, preferably an alkyl, orcombination thereof, and M is a cation which forms a water soluble saltwith the alkyl ester sulfonate. Suitable salt-forming cations includemetals such as sodium, potassium, and lithium, and substituted orunsubstituted ammonium cations, such as monoethanolamine,diethonolamine, and triethanolamine. Preferably, R³ is C₁₀-C₁₆ alkyl,and R⁴ is methyl, ethyl or isopropyl. Especially preferred are themethyl ester sulfonates wherein R³ is C₁₀-C₁₆ alkyl.

[0258] Other suitable anionic surfactants include the alkyl sulfatesurfactants which are water soluble salts or acids of the formula ROSO₃Mwherein R preferably is a C₁₀-C₂₄ hydrocarbyl, preferably an alkyl orhydroxyalkyl having a C₁₀-C₂₀ alkyl component, more preferably a C₁₂-C₁₈alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metalcation (e.g. sodium, potassium, lithium), or ammonium or substitutedammonium (e.g. methyl-, dimethyl-, and trimethyl ammonium cations andquaternary ammonium cations such as tetramethyl-ammonium and dimethylpiperdinium cations and quaternary ammonium cations derived fromalkylamines such as ethylamine, diethylamine, triethylamine, andmixtures thereof, and the like). Typically, alkyl chains of C₁₂-C₁₆ arepreferred for lower wash temperatures (e.g. below about 50 C.) andC₁₆-C₁₈ alkyl chains are preferred for higher wash temperatures (e.g.above about 50 C.).

[0259] Other anionic surfactants useful for detersive purposes can alsobe included in the laundry detergent compositions of the presentinvention. Theses can include salts (including, for example, sodium,potassium, ammonium, and substituted ammonium salts such as mono- di-and triethanolamine salts) of soap, C₈-C₂₂ primary or secondaryalkanesulfonates, C₈-C₂₄ olefinsulfonates, sulfonated polycarboxylicacids prepared by sulfonation of the pyrolyzed product of alkaline earthmetal citrates, e.g., as described in British patent specification No.1,082,179, C₈-C₂₄ alkylpolyglycolethersulfates (containing up to 10moles of ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerolsulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxideether sulfates, paraffin sulfonates, alkyl phosphates, isethionates suchas the acyl isethionates, N-acyl taurates, alkyl succinamates andsulfosuccinates, monoesters of sulfosuccinates (especially saturated andunsaturated C₁₂-C₁₈ monoesters) and diesters of sulfosuccinates(especially saturated and unsaturated C₆-C₁₂ diesters), acylsarcosinates, sulfates of alkylpolysaccharides such as the sulfates ofalkylpolyglucoside (the nonionic nonsulfated compounds being describedbelow), branched primary alkyl sulfates, and alkyl polyethoxycarboxylates such as those of the formula RO(CH₂CH₂O)_(k)—CH₂COO—M+wherein R is a C₈-C₂₂ alkyl, k is an integer from 1 to 10, and M is asoluble salt forming cation. Resin acids and hydrogenated resin acidsare also suitable, such as rosin, hydrogenated rosin, and resin acidsand hydrogenated resin acids present in or derived from tall oil.

[0260] Alkylbenzene sulfonates are highly preferred. Especiallypreferred are linear (straight-chain) alkyl benzene sulfonates (LAS)wherein the alkyl group preferably contains from 10 to 18 carbon atoms.

[0261] Further examples are described in “Surface Active Agents andDetergents” (Vol. I and II by Schwartz, Perrry and Berch). A variety ofsuch surfactants are also generally disclosed in U.S. Pat. No.3,929,678, (Column 23, line 58 through Column 29, line 23, hereinincorporated by reference).

[0262] When included therein, the laundry detergent compositions of thepresent invention typically comprise from about 1% to about 40%,preferably from about 3% to about 20% by weight of such anionicsurfactants.

[0263] The laundry detergent compositions of the present invention mayalso contain cationic, ampholytic, zwitterionic, and semi-polarsurfactants, as well as the nonionic and/or anionic surfactants otherthan those already described herein.

[0264] Cationic detersive surfactants suitable for use in the laundrydetergent compositions of the present invention are those having onelong-chain hydrocarbyl group. Examples of such cationic surfactantsinclude the ammonium surfactants such as alkyltrimethylammoniumhalogenides, and those surfactants having the formula:

[R²(OR³)_(y)][R⁴(OR³)_(y)]₂R⁵N+X−

[0265] wherein R² is an alkyl or alkyl benzyl group having from about 8to about 18 carbon atoms in the alkyl chain, each R³ is selected formthe group consisting of —CH₂CH₂—, —CH₂CH(CH₃)—, —CH₂CH(CH₂OH)—,—CH₂CH₂CH₂—, and mixtures thereof; each R⁴ is selected from the groupconsisting of C₁-C₄ alkyl, C₁-C₄ hydroxyalkyl, benzyl ring structuresformed by joining the two R⁴ groups, —CH₂CHOHCHOHCOR⁶CHOHCH₂OH, whereinR⁶ is any hexose or hexose polymer having a molecular weight less thanabout 1000, and hydrogen when y is not 0; R⁵ is the same as R⁴ or is analkyl chain, wherein the total number of carbon atoms or R² plus R⁵ isnot more than about 18; each y is from 0 to about 10,and the sum of they values is from 0 to about 15; and X is any compatible anion.

[0266] Highly preferred cationic surfactants are the water solublequaternary ammonium compounds useful in the present composition havingthe formula:

R₁R₂R₃R₄N⁺X⁻  (i)

[0267] wherein R₁ is C₈-C₁₆ alkyl, each of R₂, R₃ and R₄ isindependently C₁-C₄ alkyl, C₁-C₄ hydroxy alkyl, benzyl, and—(C₂H₄₀)_(x)H where x has a value from 2 to 5, and X is an anion. Notmore than one of R₂, R₃ or R₄ should be benzyl.

[0268] The preferred alkyl chain length for R₁ is C₁₂-C₁₅, particularlywhere the alkyl group is a mixture of chain lengths derived from coconutor palm kernel fat or is derived synthetically by olefin build up or OXOalcohols synthesis.

[0269] Preferred groups for R₂R₃ and R₄ are methyl and hydroxyethylgroups and the anion X may be selected from halide, methosulphate,acetate and phosphate ions.

[0270] Examples of suitable quaternary ammonium compounds of formulae(i) for use herein are:

[0271] coconut trimethyl ammonium chloride or bromide;

[0272] coconut methyl dihydroxyethyl ammonium chloride or bromide;

[0273] decyl triethyl ammonium chloride;

[0274] decyl dimethyl hydroxyethyl ammonium chloride or bromide;

[0275] C₁₂₋₁₅ dimethyl hydroxyethyl ammonium chloride or bromide;

[0276] coconut dimethyl hydroxyethyl ammonium chloride or bromide;

[0277] myristyl trimethyl ammonium methyl sulphate;

[0278] lauryl dimethyl benzyl ammonium chloride or bromide;

[0279] lauryl dimethyl (ethenoxy)₄ ammonium chloride or bromide;

[0280] choline esters (compounds of formula (i) wherein R₁ is

[0281] di-alkyl imidazolines [compounds of formula (i)].

[0282] Other cationic surfactants useful herein are also described inU.S. Pat. No. 4,228,044 and in EP 000 224.

[0283] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 25%, preferablyfrom about 1% to about 8% by weight of such cationic surfactants.

[0284] Ampholytic surfactants are also suitable for use in the laundrydetergent compositions of the present invention. These surfactants canbe broadly described as aliphatic derivatives of secondary or tertiaryamines, or aliphatic derivatives of heterocyclic secondary and tertiaryamines in which the aliphatic radical can be straight- orbranched-chain. One of the aliphatic substituents contains at leastabout 8 carbon atoms, typically from about 8 to about 18 carbon atoms,and at least one contains an anionic water-solubilizing group, e.g.carboxy, sulfonate, sulfate. See U.S. Pat. No. 3,929,678 (column 19,lines 18-35) for examples of ampholytic surfactants.

[0285] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 15%, preferablyfrom about 1% to about 10% by weight of such ampholytic surfactants.

[0286] Zwitterionic surfactants are also suitable for use in laundrydetergent compositions. These surfactants can be broadly described asderivatives of secondary and tertiary amines, derivatives ofheterocyclic secondary and tertiary amines, or derivatives of quaternaryammonium, quaternary phosphonium or tertiary sulfonium compounds. SeeU.S. Pat. NO. 3,929,678 (column 19, line 38 through column 22, line 48)for examples of zwitterionic surfactants.

[0287] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 15%, preferablyfrom about 1% to about 10% by weight of such zwitterionic surfactants.

[0288] Semi-polar nonionic surfactants are a special category ofnonionic surfactants which include water-soluble amine oxides containingone alkyl moiety of from about 10 to about 18 carbon atoms and 2moieties selected from the group consisting of alkyl groups andhydroxyalkyl groups containing from about 1 to about 3 carbon atoms;watersoluble phosphine oxides containing one alkyl moiety of from about10 to about 18 carbon atoms and 2 moieties selected from the groupconsisting of alkyl groups and hydroxyalkyl groups containing from about1 to about 3 carbon atoms; and water-soluble sulfoxides containing onealkyl moiety from about 10 to about 18 carbon atoms and a moietyselected from the group consisting of alkyl and hydroxyalkyl moieties offrom about 1 to about 3 carbon atoms.

[0289] Semi-polar nonionic detergent surfactants include the amine oxidesurfactants having the formula:

[0290] wherein R³ is an alkyl, hydroxyalkyl, or alkyl phenyl group ormixtures thereof containing from about 8 to about 22 carbon atoms; R⁴ isan alkylene or hydroxyalkylene group containing from about 2 to about 3carbon atoms or mixtures thereof; x is from 0 to about 3: and each R⁵ isan alkyl or hydroxyalkyl group containing from about 1 to about 3 carbonatoms or a polyethylene oxide group containing from about 1 to about 3ethylene oxide groups. The R⁵ groups can be attached to each other,e.g., through an oxygen or nitrogen atom, to form a ring structure.

[0291] These amine oxide surfactants in particular include C₁₀-C₁₈ alkyldimethyl amine oxides and C₈-C₁₂ alkoxy ethyl dihydroxy ethyl amineoxides.

[0292] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 15%, preferablyfrom about 1 % to about 10% by weight of such semi-polar nonionicsurfactants.

[0293] Builder System

[0294] The compositions according to the present invention may furthercomprise a builder system. Any conventional builder system is suitablefor use herein including aluminosilicate materials, silicates,polycarboxylates and fatty acids, materials such as ethylenediaminetetraacetate, metal ion sequestrants such as aminopolyphosphonates,particularly ethylenediamine tetramethylene phosphonic acid anddiethylene triamine pentamethylenephosphonic acid. Though less preferredfor obvious environmental reasons, phosphate builders can also be usedherein.

[0295] Suitable builders can be an inorganic ion exchange material,commonly an inorganic hydrated aluminosilicate material, moreparticularly a hydrated synthetic zeolite such as hydrated zeolite A, X,B, HS or MAP.

[0296] Another suitable inorganic builder material is layered silicate,e.g. SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consistingof sodium silicate (Na₂Si₂O₅).

[0297] Suitable polycarboxylates containing one carboxy group includelactic acid, glycolic acid and ether derivatives thereof as disclosed inBelgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylatescontaining two carboxy groups include the water-soluble salts ofsuccinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid,diglycollic acid, tartaric acid, tartronic acid and fumaric acid, aswell as the ether carboxylates described in German Offenle-enschrift2,446,686, and 2,446,487, U.S. Pat. No. 3,935,257 and the sulfinylcarboxylates described in Belgian Patent No. 840,623. Polycarboxylatescontaining three carboxy groups include, in particular, water-solublecitrates, aconitrates and citraconates as well as succinate derivativessuch as the carboxymethyloxysuccinates described in British Patent No.1,379,241, lactoxysuccinates described in Netherlands Application7205873, and the oxypolycarboxylate materials such as2-oxa-1,1,3-propane tricarboxylates described in British Patent No.1,387,447.

[0298] Polycarboxylates containing four carboxy groups includeoxydisuccinates disclosed in British Patent No.1,261,829,1,1,2,2,-ethanetetracarboxylates, 1,1,3,3-propane tetracarboxylates containing sulfosubstituents include the sulfosuccinate derivatives disclosed in BritishPatent Nos. 1,398,421 and 1,398,422 and in U.S. Pat. No. 3,936,448, andthe sulfonated pyrolysed citrates described in British Patent No.1,082,179, while polycarboxylates containing phosphone substituents aredisclosed in British Patent No. 1,439,000.

[0299] Alicyclic and heterocyclic polycarboxylates includecyclopentane-cis,cis-cis-tetracarboxylates, cyclopentadienidepentacarboxylates, 2,3,4,5-tetrahydrofuran-cis, cis,cis-tetracarboxylates, 2,5-tetrahydro-furan-cis, discarboxylates,2,2,5,5,-tetrahydrofuran-tetracarboxylates,1,2,3,4,5,6-hexane-hexacarboxylates and carboxymethyl derivatives ofpolyhydric alcohols such as sorbitol, mannitol and xylitol. Aromaticpolycarboxylates include mellitic acid, pyromellitic acid and thephthalic acid derivatives disclosed in British Patent No. 1,425,343.

[0300] Of the above, the preferred polycarboxylates arehydroxy-carboxylates containing up to three carboxy groups per molecule,more particularly citrates.

[0301] Preferred builder systems for use in the present compositionsinclude a mixture of a water-insoluble aluminosilicate builder such aszeolite A or of a layered silicate (SKS-6), and a water-solublecarboxylate chelating agent such as citric acid.

[0302] A suitable chelant for inclusion in the detergent composi-ions inaccordance with the invention is ethylenediamine-N,N′-disuccinic acid(EDDS) or the alkali metal, alkaline earth metal, ammonium, orsubstituted ammonium salts thereof, or mixtures thereof. Preferred EDDScompounds are the free acid form and the sodium or magnesium saltthereof. Examples of such preferred sodium salts of EDDS include Na₂EDDSand Na₄EDDS. Examples of such preferred magnesium salts of EDDS includeMgEDDS and Mg₂EDDS. The magnesium salts are the most preferred forinclusion in compositions in accordance with the invention.

[0303] Preferred builder systems include a mixture of a water-insolublealuminosilicate builder such as zeolite A, and a water solublecarboxylate chelating agent such as citric acid.

[0304] Other builder materials that can form part of the builder systemfor use in granular compositions include inorganic materials such asalkali metal carbonates, bicarbonates, silicates, and organic materialssuch as the organic phosphonates, amino polyalkylene phosphonates andamino polycarboxylates.

[0305] Other suitable water-soluble organic salts are the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated form each other bynot more than two carbon atoms.

[0306] Polymers of this type are disclosed in GB-A-1,596,756. Examplesof such salts are polyacrylates of MW 2000-5000 and their copolymerswith maleic anhydride, such copolymers having a molecular weight of from20,000 to 70,000, especially about 40,000.

[0307] Detergency builder salts are normally included in amounts of from5% to 80% by weight of the composition. Preferred levels of builder forliquid detergents are from 5% to 30%.

[0308] Enzymes

[0309] Preferred detergent compositions, in addition to the enzymepreparation of the invention, comprise other enzyme(s) which providescleaning performance and/or fabric care benefits.

[0310] Such enzymes include proteases, lipases, cutinases, amylases,cellulases, peroxidases, oxidases (e.g. laccases).

[0311] Proteases:

[0312] Any protease suitable for use in alkaline solutions can be used.Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically or geneticallymodified mutants are included. The protease may be a serine protease,preferably an alkaline microbial protease or a trypsin-like protease.Examples of alkaline proteases are subtilisins, especially those derivedfrom Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO 89/06270.

[0313] Preferred commercially available protease enzymes include thosesold under the trade names ALCALASE, SAVINASE, PRIMASE, DURAZYM, andESPERASE by Novo Nordisk A/S (Denmark), those sold under the tradenameMAXATASE, MAXACAL, MAXAPEM, PROPERASE, PURAFECT and PURAFECT OXP byGenencor International, and those sold under the tradename OPTICLEAN andOPTIMASE by Solvay Enzymes. Protease enzymes may be incorporated intothe compositions in accordance with the invention at a level of from0.00001% to 2% of enzyme protein by weight of the composition,preferably at a level of from 0.0001% to 1% of enzyme protein by weightof the composition, more preferably at a level of from 0.001% to 0.5% ofenzyme protein by weight of the composition, even more preferably at alevel of from 0.01 % to 0.2% of enzyme protein by weight of thecomposition.

[0314] Lipases:

[0315] Any lipase suitable for use in alkaline solutions can be used.Suitable lipases include those of bacterial or fungal origin. Chemicallyor genetically modified mutants are included.

[0316] Examples of useful lipases include a Humicola lanuginosa lipase,e.g., as described in EP 258 068 and EP 305 216, a Rhizomucor mieheilipase, e.g., as described in EP 238 023, a Candida lipase, such as a C.antarctica lipase, e.g., the C. antarctica lipase A or B described in EP214 761, a Pseudomonas lipase such as a P. alcaligenes and P.pseudoalcaligenes lipase, e.g., as described in EP 218 272, a P. cepacialipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., asdisclosed in GB 1,372,034, a P. fluorescens lipase, a Bacillus lipase,e.g., a B. subtilis lipase (Dartois et al., (1993), Biochemica etBiophysica acta 1131, 253-260), a B. stearothermophilus lipase (JP64/744992) and a B. pumilus lipase (WO 91/16422).

[0317] Furthermore, a number of cloned lipases may be useful, includingthe Penicillium camembertii lipase described by Yamaguchi et al.,(1991), Gene 103, 61-67), the Geotricum candidum lipase (Schimada, Y. etal., (1989), J. Biochem., 106, 383-388), and various Rhizopus lipasessuch as a R. delemar lipase (Hass, M. J et al., (1991), Gene 109,117-113), a R. niveus lipase (Kugimiya et al., (1992), Biosci. Biotech.Biochem. 56, 716-719) and a R. oryzae lipase.

[0318] Other types of lipolytic enzymes such as cutinases may also beuseful, e.g., a cutinase derived from Pseudomonas mendocina as describedin WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g.described in WO 90/09446).

[0319] Especially suitable lipases are lipases such as M1 Lipase™, Lumafast™ and Lipomax™ (Genencor), Lipolase™ and Lipolase Ultra™ (NovoNordisk A/S), and Lipase P “Amano” (Amano Pharmaceutical Co. Ltd.).

[0320] The lipases are normally incorporated in the detergentcomposition at a level of from 0.00001% to 2% of enzyme protein byweight of the composition, preferably at a level of from 0.0001 % to 1 %of enzyme protein by weight of the composition, more preferably at alevel of from 0.001 % to 0.5% of enzyme protein by weight of thecomposition, even more preferably at a level of from 0.01 % to 0.2% ofenzyme protein by weight of the composition.

[0321] Amylases:

[0322] Any amylase (alpha and/or beta) suitable for use in alkalinesolutions can be used. Suitable amylases include those of bacterial orfungal origin. Chemically or genetically modified mutants are included.Amylases include, for example, alpha-amylases obtained from a specialstrain of B. licheniformis, described in more detail in GB 1,296,839.Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (available from Novo Nordisk A/S) and Rapidase™ and Maxamyl P™(available from Genencor).

[0323] The amylases are normally incorporated in the detergentcomposition at a level of from 0.00001% to 2% of enzyme protein byweight of the composition, preferably at a level of from 0.0001 % to 1 %of enzyme protein by weight of the composition, more preferably at alevel of from 0.001 % to 0.5% of enzyme protein by weight of thecomposition, even more preferably at a level of from 0.01 % to 0.2% ofenzyme protein by weight of the composition.

[0324] Cellulases:

[0325] Any cellulase suitable for use in alkaline solutions can be used.Suitable cellulases include those of bacterial or fungal origin.Chemically or genetically modified mutants are included. Suitablecellulases are disclosed in U.S. Pat. No. 4,435,307, which disclosesfungal cellulases produced from Humicola insolens. Especially suitablecellulases are the cellulases having colour care benefits. Examples ofsuch cellulases are cellulases described in European patent applicationNo. 0 495 257.

[0326] Commercially available cellulases include Celluzyme produced by astrain of Humicola insolens, (Novo Nordisk A/S), and KAC-500(B) (KaoCorporation).

[0327] Cellulases are normally incorporated in the detergent compositionat a level of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001 % to 0.5% of enzyme protein by weight of the composition, evenmore preferably at a level of from 0.01 % to 0.2% of enzyme protein byweight of the composition.

[0328] Peroxidases/Oxidases:

[0329] Peroxidase enzymes are used in combination with hydrogen peroxideor a source thereof (e.g. a percarbonate, perborate or persulfate).Oxidase enzymes are used in combination with oxygen. Both types ofenzymes are used for “solution bleaching”, i.e. to prevent transfer of atextile dye from a dyed fabric to another fabric when said fabrics arewashed together in a wash liquor, preferably together with an enhancingagent as described in e.g. WO 94/12621 and WO 95/01426. Suitableperoxidases/oxidases include those of plant, bacterial or fungal origin.Chemically or genetically modified mutants are included.

[0330] Peroxidase and/or oxidase enzymes are normally incorporated inthe detergent composition at a level of from 0.00001 % to 2% of enzymeprotein by weight of the composition, preferably at a level of from0.0001 % to 1 % of enzyme protein by weight of the composition, morepreferably at a level of from 0.001 % to 0.5% of enzyme protein byweight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.

[0331] Mixtures of the above mentioned enzymes are encompassed herein,in particular a mixture of a protease, an amylase, a lipase and/or acellulase.

[0332] The enzyme of the invention, or any other enzyme incorporated inthe detergent composition, is normally incorporated in the detergentcomposition at a level from 0.00001 % to 2% of enzyme protein by weightof the composition, preferably at a level from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level from 0.01 % to 0.2% of enzyme protein by weight ofthe composition.

[0333] Bleaching Agents:

[0334] Additional optional detergent ingredients that can be included inthe detergent compositions of the present invention include bleachingagents such as PB1, PB4 and percarbonate with a particle size of 400-800microns. These bleaching agent components can include one or more oxygenbleaching agents and, depending upon the bleaching agent chosen, one ormore bleach activators. When present oxygen bleaching compounds willtypically be present at levels of from about 1 % to about 25%. Ingeneral, bleaching compounds are optional added components in non-liquidformulations, e.g. granular detergents.

[0335] The bleaching agent component for use herein can be any of thebleaching agents useful for detergent compositions including oxygenbleaches as well as others known in the art.

[0336] The bleaching agent suitable for the present invention can be anactivated or non-activated bleaching agent.

[0337] One category of oxygen bleaching agent that can be usedencompasses percarboxylic acid bleaching agents and salts thereof.Suitable examples of this class of agents include magnesiummonoperoxyphthalate hexahydrate, the magnesium salt of meta-chloroperbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid anddiperoxydodecanedioic acid. Such bleaching agents are disclosed in U.S.Pat. No. 4,483,781, U.S. Pat. No. 740,446, EP 0 133 354 and U.S. Pat.No. 4,412,934. Highly preferred bleaching agents also include6-nonylamino-6-oxoperoxycaproic acid as described in U.S. Pat. No.4,634,551.

[0338] Another category of bleaching agents that can be used encompassesthe halogen bleaching agents. Examples of hypohalite bleaching agents,for example, include trichloro isocyanuric acid and the sodium andpotassium dichloroisocyanurates and N-chloro and N-bromo alkanesulphonamides. Such materials are normally added at 0.5-10% by weight ofthe finished product, preferably 1-5% by weight.

[0339] The hydrogen peroxide releasing agents can be used in combinationwith bleach activators such as tetra-acetylethylenediamine (TAED),nonanoyloxybenzenesulfonate (NOBS, described in U.S. Pat. No.4,412,934), 3,5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS,described in EP 120 591) or pentaacetylglucose (PAG), which areperhydrolyzed to form a peracid as the active bleaching species, leadingto improved bleaching effect. In addition, very suitable are the bleachactivators C8(6-octanamido-caproyl) oxybenzene-sulfonate,C9(6-nonanamido caproyl) oxybenzenesulfonate and C10 (6-decanamidocaproyl) oxybenzenesulfonate or mixtures thereof. Also suitableactivators are acylated citrate esters such as disclosed in EuropeanPatent Application No. 91870207.7.

[0340] Useful bleaching agents, including peroxyacids and bleachingsystems comprising bleach activators and peroxygen bleaching compoundsfor use in cleaning compositions according to the invention aredescribed in application U.S. Ser. No. 08/136,626.

[0341] The hydrogen peroxide may also be present by adding an enzymaticsystem (i.e. an enzyme and a substrate therefore) which is capable ofgeneration of hydrogen peroxide at the beginning or during the washingand/or rinsing process. Such enzymatic systems are disclosed in EuropeanPatent Application EP 0 537 381.

[0342] Bleaching agents other than oxygen bleaching agents are alsoknown in the art and can be utilized herein. One type of non-oxygenbleaching agent of particular interest includes photoactivated bleachingagents such as the sulfonated zinc and/or aluminium phthalocyanines.These materials can be deposited upon the substrate during the washingprocess. Upon irradiation with light, in the presence of oxygen, such asby hanging clothes out to dry in the daylight, the sulfonated zincphthalocyanine is activated and, consequently, the substrate isbleached. Preferred zinc phthalocyanine and a photoactivated bleachingprocess are described in U.S. Pat. No. 4,033,718. Typically, detergentcomposition will contain about 0.025% to about 1.25%, by weight, ofsulfonated zinc phthalocyanine.

[0343] Bleaching agents may also comprise a manganese catalyst. Themanganese catalyst may, e.g., be one of the compounds described in“Efficient manganese catalysts for low-temperature bleaching”, Nature369, 1994, pp. 637-639.

[0344] Suds Suppressors:

[0345] Another optional ingredient is a suds suppressor, exemplified bysilicones, and silica-silicone mixtures. Silicones can generally berepresented by alkylated polysiloxane materials, while silica isnormally used in finely divided forms exemplified by silica aerogels andxerogels and hydrophobic silicas of various types. Theses materials canbe incorporated as particulates, in which the suds suppressor isadvantageously releasably incorporated in a water-soluble orwaterdispersible, substantially non surface-active detergent impermeablecarrier. Alternatively the suds suppressor can be dissolved or dispersedin a liquid carrier and applied by spraying on to one or more of theother components.

[0346] A preferred silicone suds controlling agent is disclosed in U.S.Pat. No. 3,933,672. Other particularly useful suds suppressors are theself-emulsifying silicone suds suppressors, described in German PatentApplication DTOS 2,646,126. An example of such a compound is DC-544,commercially available form Dow Corning, which is a siloxane-glycolcopolymer. Especially preferred suds controlling agent are the sudssuppressor system comprising a mixture of silicone oils and2-alkyl-alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanol whichare commercially available under the trade name Isofol 12 R.

[0347] Such suds suppressor system are described in European PatentApplication EP 0 593 841.

[0348] Especially preferred silicone suds controlling agents aredescribed in European Patent Application No. 92201649.8. Saidcompositions can comprise a silicone/silica mixture in combination withfumed nonporous silica such as Aerosil^(R).

[0349] The suds suppressors described above are normally employed atlevels of from 0.001 % to 2% by weight of the composition, preferablyfrom 0.01 % to 1 % by weight.

[0350] Other Components:

[0351] Other components used in detergent compositions may be employedsuch as soil-suspending agents, soil-releasing agents, opticalbrighteners, abrasives, bactericides, tarnish inhibitors, coloringagents, and/or encapsulated or nonencapsulated perfumes.

[0352] Especially suitable encapsulating materials are water solublecapsules which consist of a matrix of polysaccharide and polyhydroxycompounds such as described in GB 1,464,616.

[0353] Other suitable water soluble encapsulating materials comprisedextrins derived from ungelatinized starch acid esters of substituteddicarboxylic acids such as described in U.S. Pat. No. 3,455,838. Theseacid-ester dextrins are, preferably, prepared from such starches as waxymaize, waxy sorghum, sago, tapioca and potato. Suitable examples of saidencapsulation materials include N-Lok manufactured by National Starch.The N-Lok encapsulating material consists of a modified maize starch andglucose. The starch is modified by adding monofunctional substitutedgroups such as octenyl succinic acid anhydride.

[0354] Antiredeposition and soil suspension agents suitable hereininclude cellulose derivatives such as methylcellulose,carboxymethylcellulose and hydroxyethylcellulose, and homo- orco-polymeric polycarboxylic acids or their salts. Polymers of this typeinclude the polyacrylates and maleic anhydride-acrylic acid copolymerspreviously mentioned as builders, as well as copolymers of maleicanhydride with ethylene, methylvinyl ether or methacrylic acid, themaleic anhydride constituting at least 20 mole percent of the copolymer.These materials are normally used at levels of from 0.5% to 10% byweight, more preferably form 0.75% to 8%, most preferably from 1 % to 6%by weight of the composition.

[0355] Preferred optical brighteners are anionic in character, examplesof which are disodium 4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2:2′disulphonate, disodium4,4′-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino-stilbene-2:2′-disulphonate,disodium4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2:2′-disulphonate,monosodium4′,4″-bis-(2,4-dianilino-s-tri-azin-6-ylamino)stilbene-2-sulphonate,disodium4,4′-bis-(2-anilino-4-(N-methyl-N-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,di-sodium4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2′disulphonate,di-sodium4,4′-bis(2-anilino-4-(1-methyl-2-hydroxyethylamino)-s-triazin-6-ylami-no)stilbene-2,2′disulphonate,sodium 2(stilbyl-4″-(naphtho-1′, 2′:4,5)-1,2,3-triazole-2″-sulphonateand 4,4′-bis(2-sulphostyryl)biphenyl.

[0356] Other useful polymeric materials are the polyethylene glycols,particularly those of molecular weight 1000-10000, more particularly2000 to 8000 and most preferably about 4000. These are used at levels offrom 0.20% to 5% more preferably from 0.25% to 2.5% by weight. Thesepolymers and the previously mentioned homo- or co-polymericpoly-carboxylate salts are valuable for improving whiteness maintenance,fabric ash deposition, and cleaning performance on clay, proteinaceousand oxidizable soils in the presence of transition metal impurities.

[0357] Soil release agents useful in compositions of the presentinvention are conventionally copolymers or terpolymers of terephthalicacid with ethylene glycol and/or propylene glycol units in variousarrangements. Examples of such polymers are disclosed in U.S. Pat. No.4,116,885 and 4,711,730 and EP 0 272 033. A particular preferred polymerin accordance with EP 0 272 033 has the formula:

(CH₃(PEG)₄₃)_(0.75)(POH)_(0.25)[T-PO)_(2.8)(T-PEG)_(0.4)]T(POH)_(0.25)((PEG)₄₃CH₃)_(0.75)

[0358] where PEG is —(OC₂H₄)0-, PO is (OC₃H₆O) and T is (POOC₆H₄CO).

[0359] Also very useful are modified polyesters as random copolymers ofdimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and1,2-propanediol, the end groups consisting primarily of sulphobenzoateand secondarily of mono esters of ethylene glycol and/or1,2-propanediol. The target is to obtain a polymer capped at both end bysulphobenzoate groups, “primarily”, in the present context most of saidcopolymers herein will be endcapped by sulphobenzoate groups. However,some copolymers will be less than fully capped, and therefore their endgroups may consist of monoester of ethylene glycol and/or1,2-propanediol, thereof consist “secondarily” of such species.

[0360] The selected polyesters herein contain about 46% by weight ofdimethyl terephthalic acid, about 16% by weight of 1,2-propanediol,about 10% by weight ethylene glycol, about 13% by weight of dimethylsulfobenzoic acid and about 15% by weight of sulfoisophthalic acid, andhave a molecular weight of about 3.000. The polyesters and their methodof preparation are described in detail in EP 311 342.

[0361] Softening Agents:

[0362] Fabric softening agents can also be incorporated into laundrydetergent compositions in accordance with the present invention. Theseagents may be inorganic or organic in type. Inorganic softening agentsare exemplified by the smectite clays disclosed in GB-A-1 400898 and inU.S. Pat. No. 5,019,292. Organic fabric softening agents include thewater insoluble tertiary amines as disclosed in GB-A1 514 276 and EP 0011 340 and their combination with mono C₁₂-C₁₄ quaternary ammoniumsalts are disclosed in EP-B-0 026 528 and di-long-chain amides asdisclosed in EP 0 242 919. Other useful organic ingredients of fabricsoftening systems include high molecular weight polyethylene oxidematerials as disclosed in EP 0 299 575 and 0 313 146.

[0363] Levels of smectite clay are normally in the range from 5% to 15%,more preferably from 8% to 12% by weight, with the material being addedas a dry mixed component to the remainder of the formulation. Organicfabric softening agents such as the water-insoluble tertiary amines ordilong chain amide materials are incorporated at levels of from 0.5% to5% by weight, normally from 1 % to 3% by weight whilst the highmolecular weight polyethylene oxide materials and the water solublecationic materials are added at levels of from 0.1 % to 2%, normallyfrom 0.15% to 1.5% by weight. These materials are normally added to thespray dried portion of the composition, although in some instances itmay be more convenient to add them as a dry mixed particulate, or spraythem as molten liquid on to other solid components of the composition.

[0364] Polymeric Dye-transfer Inhibiting Agents:

[0365] The detergent compositions according to the present invention mayalso comprise from 0.001 % to 10%, preferably from 0.01 % to 2%, morepreferably form 0.05% to 1% by weight of polymeric dye-transferinhibiting agents. Said polymeric dye-transfer inhibiting agents arenormally incorporated into detergent compositions in order to inhibitthe transfer of dyes from colored fabrics onto fabrics washed therewith.These polymers have the ability of complexing or adsorbing the fugitivedyes washed out of dyed fabrics before the dyes have the opportunity tobecome attached to other articles in the wash.

[0366] Especially suitable polymeric dye-transfer inhibiting agents arepolyamine N-oxide polymers, copolymers of N-vinyl-pyrrolidone andN-vinylimidazole, polyvinylpyrrolidone polymers, polyvinyloxazolidonesand polyvinylimidazoles or mixtures thereof.

[0367] Addition of such polymers also enhances the performance of theenzymes according the invention.

[0368] The detergent composition according to the invention can be inliquid, paste, gels, bars or granular forms.

[0369] Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) andmay optionally be coated by methods known in the art. Examples of waxycoating materials are poly(ethylene oxide) products (polyethyleneglycol,PEG) with mean molecular weights of 1000 to 20000; ethoxylatednonylphenols having from 16 to 50 ethylene oxide units; ethoxylatedfatty alcohols in which the alcohol contains from 12 to 20 carbon atomsand in which there are 15 to 80 ethylene oxide units; fatty alcohols;fatty acids; and mono- and di- and triglycerides of fatty acids.Examples of film-forming coating materials suitable for application byfluid bed techniques are given in GB 1483591.

[0370] Granular compositions according to the present invention can alsobe in “compact form”, i.e. they may have a relatively higher densitythan conventional granular detergents, i.e. form 550 to 950 g/l; in suchcase, the granular detergent compositions according to the presentinvention will contain a lower amount of “Inorganic filler salt”,compared to conventional granular detergents; typical filler salts arealkaline earth metal salts of sulphates and chlorides, typically sodiumsulphate; “Compact” detergent typically comprise not more than 10%filler salt. The liquid compositions according to the present inventioncan also be in “concentrated form”, in such case, the liquid detergentcompositions according to the present invention will contain a loweramount of water, compared to conventional liquid detergents. Typically,the water content of the concentrated liquid detergent is less than 30%,more preferably less than 20%, most preferably less than 10% by weightof the detergent compositions.

[0371] The compositions of the invention may for example, be formulatedas hand and machine laundry detergent compositions including laundryadditive compositions and compositions suitable for use in thepretreatment of stained fabrics, rinse added fabric softenercompositions, and compositions for use in general household hard surfacecleaning operations and dishwashing operations.

[0372] The following examples are meant to exemplify compositions forthe present invention, but are not necessarily meant to limit orotherwise define the scope of the invention.

[0373] In the detergent compositions, the abbreviated componentidentifications have the following meanings: LAS: Sodium linear C₁₂alkyl benzene sulphonate TAS: Sodium tallow alkyl sulphate XYAS: SodiumC_(1x)-C_(1Y) alkyl sulfate SS: Secondary soap surfactant of formula2-butyl octanoic acid 25EY: A C₁₂-C₁₅ predominantly linear primaryalcohol condensed with an average of Y moles of ethylene oxide 45EY: AC₁₄-C₁₅ predominantly linear primary alcohol condensed with an averageof Y moles of ethylene oxide XYEZS: C_(1x)-C_(1Y) sodium alkyl sulfatecondensed with an average of Z moles of ethylene oxide per moleNonionic: C₁₃-C₁₅ mixed ethoxylated/propoxylated fatty alcohol with anaverage degree of ethoxylation of 3.8 and an average degree ofpropoxylation of 4.5 sold under the tradename Plurafax LF404 by BASFGmbh CFAA: C₁₂-C₁₄ alkyl N-methyl glucamide TFAA: C₁₆-C₁₈ alkyl N-methylglucamide Silicate: Amorphous Sodium Silicate (SiO₂:Na₂O ratio = 2.0)NaSKS-6: Crystalline layered silicate of formula delta- Na₂Si₂O₅Carbonate: Anhydrous sodium carbonate Phosphate: Sodium tripolyphosphateMA/AA: Copolymer of 1:4 maleic/acrylic acid, average molecular weightabout 80,000 Polyacrylate: Polyacrylate homopolymer with an averagemolecular weight of 8,000 sold under the tradename PA30 by BASF GmbhZeolite A: Hydrated Sodium Aluminosilicate of formulaNa₁₂(AlO₂SiO₂)₁₂.27H₂O having a primary particle size in the range from1 to 10 micrometers Citrate: Tri-sodium citrate dihydrate Citric: CitricAcid Perborate: Anhydrous sodium perborate monohydrate bleach, empiricalformula NaBO₂.H₂O₂ PB4: Anhydrous sodium perborate tetrahydratePercarbonate: Anhydrous sodium percarbonate bleach of empirical formula2Na₂CP₃.3H₂O₂ TAED: Tetraacetyl ethylene diamine CMC: Sodiumcarboxymethyl cellulose DETPMP: Diethylene triamine penta (methylenephosphonic acid), marketed by Monsanto under the Tradename Dequest 2060PVP: Polyvinylpyrrolidone polymer EDDS: Ethylenediamine-N,N′-disuccinicacid, [5,5] isomer in the form of the sodium salt Suds 25% paraffin waxMpt 50° C., 17% hydrophobic silica, 58% Suppressor: paraffin oilGranular Suds 12% Silicone/silica, 18% stearyl alcohol, 70% suppressor:starch in granular form Sulphate: Anhydrous sodium sulphate HMWPEO: Highmolecular weight polyethylene oxide TAE 25: Tallow alcohol ethoxylate(25)

DETERGENT EXAMPLE I

[0374] A granular fabric cleaning composition in accordance with theinvention may be prepared as follows: Sodium linear C₁₂ alkyl 6.5benzene sulfonate Sodium sulfate 15.0 Zeolite A 26.0 Sodiumnitrilotriacetate 5.0 Enzyme of the invention 0.1 PVP 0.5 TAED 3.0 Boricacid 4.0 Perborate 18.0 Phenol sulphonate 0.1 Minors Up to 100

DETERGENT EXAMPLE II

[0375] A compact granular fabric cleaning composition (density 800 g/l)in accord with the invention may be prepared as follows: 45AS 8.0 25E3S2.0 25E5 3.0 25E3 3.0 TFAA 2.5 Zeolite A 17.0 NaSKS-6 12.0 Citric acid3.0 Carbonate 7.0 MA/AA 5.0 CMC 0.4 Enzyme of the invention 0.1 TAED 6.0Percarbonate 22.0 EDDS 0.3 Granular suds suppressor 3.5 water/minors Upto 100%

DETERGENT EXAMPLE III

[0376] Granular fabric cleaning compositions in accordance with theinvention which are especially useful in the laundering of colouredfabrics were prepared as follows: LAS 10.7 — TAS 2.4 — TFAA — 4.0 45AS3.1 10.0 45E7 4.0 — 25E3S — 3.0 68E11 1.8 — 25E5 — 8.0 Citrate 15.0 7.0Carbonate — 10.0 Citric acid 2.5 3.0 Zeolite A 32.1 25.0 Na-SKS-6 — 9.0MNAA 5.0 5.0 DETPMP 0.2 0.8 Enzyme of the invention 0.10 0.05 Silicate2.5 — Sulphate 5.2 3.0 PVP 0.5 — Poly (4-vinylpyridine)-N- — 0.2Oxide/copolymer of vinyl- imidazole and vinyl- pyrrolidone Perborate 1.0— Phenol sulfonate 0.2 — Water/Minors Up to 100%

DETERGENT EXAMPLE IV

[0377] Granular fabric cleaning compositions in accordance with theinvention which provide “Softening through the wash” capability may beprepared as follows: 45AS — 10.0 LAS 7.6 — 68AS 1.3 — 45E7 4.0 — 25E3 —5.0 Coco-alkyl-dimethyl hydro- 1.4 1.0 xyethyl ammonium chloride Citrate5.0 3.0 Na-SKS-6 — 11.0 Zeolite A 15.0 15.0 MA/AA 4.0 4.0 DETPMP 0.4 0.4Perborate 15.0 — Percarbonate — 15.0 TAED 5.0 5.0 Smectite clay 10.010.0 HMWPEO — 0.1 Enzyme of the invention 0.10 0.05 Silicate 3.0 5.0Carbonate 10.0 10.0 Granular suds suppressor 1.0 4.0 CMC 0.2 0.1Water/Minors Up to 100%

DETERGENT EXAMPLE V

[0378] Heavy duty liquid fabric cleaning compositions in accordance withthe invention may be prepared as follows: I II LAS acid form — 25.0Citric acid 5.0 2.0 25AS acid form 8.0 — 25AE2S acid form 3.0 — 25AE78.0 — CFAA 5 — DETPMP 1.0 1.0 Fatty acid 8 — Oleic acid — 1.0 Ethanol4.0 6.0 Propanediol 2.0 6.0 Enzyme of the invention 0.10 0.05 Coco-aikyldimethyl — 3.0 hydroxy ethyl ammonium chloride Smectite clay — 5.0 PVP2.0 — Waterl Minors Up to 100%

[0379] Materials and Methods

[0380] Strains:

[0381]B. lentus 309 and 147 are specific strains of Bacillus lentus,deposited with the NCIB and accorded the accession numbers NCIB 10309and 10147, and described in U.S. Pat. No. 3,723,250 incorporated byreference herein.

[0382]E. coli MC₁₀₀₀ (M. J. Casadaban and S. N. Cohen (1980); J. Mol.Biol. 138 179-207), was made r⁻,m⁺ by conventional methods and is alsodescribed in U.S. patent application Ser. No. 039,298.

[0383] Plasmids:

[0384] pJS3: E. coli-B. subtilis shuttle vector containing a syntheticgene encoding for subtilase 309. (Described by Jacob Schiødt et al. inProtein and Peptide letters 3:39-44 (1996)).

[0385] pSX222: B. subtilis expression vector (Described inPCT/DK96/00207).

[0386] General Molecular Biology Methods:

[0387] Unless otherwise mentioned the DNA manipulations andtransformations were performed using standard methods of molecularbiology (Sambrook et al. (1989) Molecular cloning: A laboratory manual,Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al.(eds.) “Current protocols in Molecular Biology”. John Wiley and Sons,1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular BiologicalMethods for Bacillus”. John Wiley and Sons, 1990).

[0388] Enzymes for DNA manipulations were used according to thespecifications of the suppliers.

[0389] Enzymes for DNA Manipulations

[0390] Unless otherwise mentioned all enzymes for DNA manipulations,such as e.g. restiction endonucleases, ligases etc., are obtained fromNew England Biolabs, Inc.

[0391] Construction of Random Mutagenized Libraries

[0392] Performing Localized Random Mutagenesis

[0393] A mutagenic primer (oligonucleotide) is synthesized whichcorresponds to the part of the DNA sequence to be mutagenized except forthe nucleotide(s) corresponding to amino acid codon(s) to bemutagenized.

[0394] Subsequently, the resulting mutagenic primer is used in a PCRreaction with a suitable opposite primer. The resulting PCR fragment ispurified and digested and cloned into the shuttle vector.

[0395] Alternatively and if necessary, the resulting PCR fragment isused in a second PCR reaction as a primer with a second suitableopposite primer so as to allow digestion and cloning of the mutagenizedregion into the shuttle vector. The PCR reactions are performed undernormal conditions.

[0396] Proteolytic Activity

[0397] In the context of this invention proteolytic activity isexpressed in Kilo NOVO Protease Units (KNPU). The activity is determinedrelatively to an enzyme standard (SAVINASE®), and the determination isbased on the digestion of a dimethyl casein (DMC) solution by theproteolytic enzyme at standard conditions, i.e. 50° C., pH 8.3, 9 min.reaction time, 3 min. measuring time. A folder AF 220/1 is availableupon request to Novo Nordisk A/S, Denmark, which folder is herebyincluded by reference.

[0398] A GU is a Glycine Unit, defined as the proteolytic enzymeactivity which, under standard conditions, during a 15-minutes'incubation at 40° C., with N-acetyl casein as substrate, produces anamount of NH₂-group equivalent to 1 mmole of glycine.

[0399] Enzyme activity can also be measured using the PNA assay,according to reaction with the soluble substratesuccinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, whichis described in the Journal of American Oil Chemists Society, Rothgeb,T. M., Goodlander, B. D., Garrison, P. H., and Smith, L. A., (1988).

[0400] Fermentation:

[0401] Fermentation of subtilase enzymes was performed at 30° C. on arotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flaskscontaining 100 ml BPX medium for 5 days.

[0402] Consequently, in order to make an e.g. 2 liter broth 20Erlenmeyer flasks were fermented simultaneously.

[0403] Assay to Test for Proteases with Increased AutoproteolyticStability:

[0404] Samples containing protease are made by growing strains includingreference strains in suitable media allowing expression of protease ineither microtiter plates or shake flasks.

[0405] From each sample containing protease, aliquots are taken, towhich are added 1/10 vol.2 M Glycin-NaOH buffer; pH 10.0, and thealiquots are incubated for 3 hours at 4° C. and 55° C., respectively.

[0406] After incubation the protease activities are determined using thesubstrate succinyl-alanine-alanine-para-nitrophenol (Suc-Ala-Ala-pNA) at0.6 g/l in the following buffer:

[0407] 150 mM KCl, 50 mM Na₂B₄O₇; pH adjusted to 9.0.

[0408] 20 micro-I sample+180 micro-I substrate are mixed in wells of96-well microtiter plate.

[0409] Color development is followed at 405 nm in a microplate reader.

[0410] Activities are determined using SAVINASE® as standard.

[0411] The residual activity of a sample is calculated as the proteaseactivity of the aliquot incubated at 55° C. in percentage of theprotease activity of the aliquot incubated at 4° C.

[0412] The proteases exhibiting increased residual activity areidentified.

[0413] Media:

[0414] BPX: Composition (per Liter) Potato starch 100 g Ground barley 50g Soybean flour 20 g Na₂HPO₄X 12 H₂O 9 g Pluronic 0.1 g Sodium caseinate10 g

[0415] The starch in the medium is liquefied with alpha-amylase and themedium is sterilized by heating at 120° C. for 45 minutes. Aftersterilization the pH of the medium is adjusted to 9 by addition ofNaHCO₃ to 0.1 M.

EXAMPLES

[0416] For the generation of enzyme variants according to the inventionthe same materials and methods as described in i.a. WO 89/06279 (NovoNordisk A/S), EP 130,756 (Genentech), EP 479,870 (Novo Nordisk A/S), EP214,435 (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EPapplication no. 87303761 (Genentech), EP 260,105 (Genencor), WO 88/06624(Gist-Brocades NV), WO 88/07578 (Genentech), WO 88/08028 (Genex), WO88/08033 (Amgen), WO 88/08164 (Genex), Thomas et al. (1985) Nature, 318375-376; Thomas et al. (1987) J. Mol. Biol., 193, 803-813; Russel andFersht (1987) Nature 328 496-500. Other methods well established in theart may also be used.

Example 1

[0417] Construction and Expression of Enzyme Variants:

[0418] Subtilase 309 site-directed variants were made by the “Uniquesite elimination (USE)” or the “Uracil-USE” technique describedrespectively by Deng et al. (Anal. Biochem. 200:81-88 (1992)) andMarkvardsen et al. (BioTechniques 18(3):371-372 (1995)).

[0419] The template plasmid was pJS3, or an analogue of this containinga variant of Subtilase 309, e.g. USE mutagenesis was performed on pJS3analogue containing a gene encoding the Y167I+R170L variant with aoligonucleotide directed to the construct A194P variant resulting in afinal Y167I+R170L+A194P Subtilase 309 variant.

[0420] The in pJS3 constructed Subtilase 309 variants was then subclonedinto the B. subtilis pSX222 expression plasmid, using the restrictionenzymes KpnI and MluI.

[0421] This construct was transformed into a competent B. subtilisstrain and in order to purify the protease fermented as described abovein a medium containing 10 microg/ml Chloramphenicol (CAM).

Example 2

[0422] Purification of Enzyme Variants:

[0423] This procedure relates to purification of a 2 liter scalefermentation of the Subtilisin 147 enzyme, the Subtilisin 309 enzyme ormutants thereof.

[0424] Approximately 1.6 liters of fermentation broth were centrifugedat 5000 rpm for 35 minutes in 1 liter beakers. The supernatants wereadjusted to pH 6.5 using 10% acetic acid and filtered on Seitz SupraS100 filter plates.

[0425] The filtrates were concentrated to approximately 400 ml using anAmicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UFconcentrate was centrifuged and filtered prior to absorption at roomtemperature on a Bacitracin affinity column at pH 7. The protease waseluted from the Bacitracin column at room temperature using 25%2-propanol and 1 M sodium chloride in a buffer solution with 0.01dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chlorideadjusted to pH 7.

[0426] The fractions with protease activity from the Bacitracinpurification step were combined and applied to a 750 ml Sephadex G25column (5 cm dia.) equilibrated with a buffer containing 0.01dimethylglutaric acid, 0.2 M boric acid and 0.002 m calcium chlorideadjusted to pH 6.5.

[0427] Fractions with proteolytic activity from the Sephadex G25 columnwere combined and applied to a 150 ml CM Sepharose CL 6B cation exchangecolumn (5 cm dia.) equilibrated with a buffer containing 0.01 Mdimethylglutaric acid, 0.2 M boric acid, and 0.002 M calcium chlorideadjusted to pH 6.5.

[0428] The protease was eluted using a linear gradient of 0-0.1 M sodiumchloride in 2 liters of the same buffer (0-0.2 M sodium chloride in caseof Subtilisin 147).

[0429] In a final purification step protease containing fractions fromthe CM Sepharose column were combined and concentrated in an Amiconultrafiltration cell equipped with a GR81 PP membrane (from the DanishSugar Factories Inc.).

[0430] By using the techniques of Example 1 for the construction and theabove isolation procedure the following subtilisin 309 variants wereproduced and isolated: A: Y167I + R170L + A133P B: Y167I + R170L + T134PC: Y167I + R170L + A133P + T134P D: Y167I + R170L + V104C + S132C E:Y167I + R170L + A108C + T134C F: Y167A + R170S + F189A G: Y167A +R170S + Y192A H: Y167A + R170S + Y192P I: Y167A + R170S + Y192A + A194PJ: Y167A + R170S + Y192P + A194P K: Y167A + R170S + F189G L: Y167A +R170S + F189E M: Y167A + R170S + F189R N: Y167I + R170L M: Y167I +R170L + A194P O: Y167A + R170S + A194P P: Y167A + R170L + A194P Q:Y167A + R170N + A194P R: V104C + S132C + Y1671 + R170L S: A108C +T134C + Y1671 + R170L

Example 3

[0431] Identification of Autolytic Cleavage Site:

[0432] A fraction of the SAVINASE® variant N: 167I+R170L (afterpurification as described above) was by SDS-PAGE analysis found tocontain two bands supposedly originating from autolytic degradation. Thebands migrated with Mr's of 12 kDa and 10 kDa, respectively. TheN-terminal amino acid sequences of the peptides constituting these twobands were determined following SDS-PAGE and electroblotting onto a PVDFmembrane.

[0433] The N-terminal amino acid sequence of the band migrating with Mr12 kDa was found to be Ala-Gln-Ser-Val-Pro-Trp-Gly-lle-Ser- which isidentical to the N-terminal amino acid sequence of N:Y167I+R170L.

[0434] The N-terminal amino acid sequence of the band migrating with Mr10 kDa was found to be Gly-Ala-Gly-Leu-Asp-Ile-Val-Ala-Pro- which isidentical to the amino acid sequence of amino acid residues 187 to 195in N:Y167I+R170L (residues 193 to 201 in BPN′ numbering). Thisidentifies the peptide bond between amino acid residues 186 and 187 inN:Y167I+R170L (residues 192 and 193 in BPN′ numbering) as an autolyticcleavage site.

[0435] Matrix assisted laser desorption ionisation time-of-flight massspectrometry of the fraction revealed that the masses of the twocomponents were 12,997.5 KDa±13 KDa and 8,397.8 KDa±8 KDa, respectively.

[0436] The theoretical mass of the autolytic fragment consisting ofamino acid residues 187 to 269 in N:Y167I+R170L (residues 193 to 275 inBPN′ numbering) is 8,397.3 KDa thereby confirming that no otherautolytic cleavages have occured C-terminal to residue 186.

[0437] Using the mass value of the larger fragment (12,997.5 KDa) it ispossible to deduce that the other autolytic cleavage has occured betweenamino acid residues 130 and 131 in N:Y167I+R170L (residues 132 and 133in BPN′ numbering). The theoretical mass of the autolytic fragmentconsisting of amino acid residues 1 to 130 in N:Y167I+R170L (residues 1to 132 in BPN′ numbering) is 12,699.1 kDa.

[0438] To substantiate these findings of autolytic cleavage sites inN:Y167I+R170L, the protease was incubated in a concentration of 2 mg/mlin 0.1 M sodium phosphate, pH 7.5 at 37° C. At various time points from0 min to 6 hours a 20 microliter aliqout of the incubation mixture waswithdrawn and added 80 microliters 1% TFA resulting in an irreversibleinhibition of the proteolytic activity of N:Y167I+R170L. The sampleswere kept in the freezer until analysis by matrix assisted laserdesorption time-of-flight mass spectrometry.

[0439] The result of the mass spectrometry clearly showed a steadyincrease in the amount of fragments with masses of 12,698.9 kDa±13 kDaand 8,396.1 kDa±8 kDa, respectively, and a steady decrease in thecomponent with mass 26,607.0 KDa±26 kDa. The theoretical mass ofN:Y167I+R170L is 26,605.4 kDa.

Example 4

[0440] Construction of Random Protease Variants

[0441] Three random libraries was constructed in the vicinity of theautoproteolytic claevage site 132-133. In each of the 3 libraries theBLS309 variant Y167I+R170L was used as template.

[0442] The construction of the 3 libraries of amino acids (aa) 1)129-131, 2)132-133, 3)134-135 were prepared as described in Materialsand Methods above.

[0443] One oligonucleotide was synthesized with 25% of each of the fourbases in the first and the second base at amino acid codons wanted to bemutagenized. The third nucleotide (the wobble base) in codons weresynthesized with 50%G/50%C to give a larger likelihood for changes toamino acids with one or two codons.

[0444] The mutagenic primer was used in a PCR reaction with a suitableopposite primer and the plasmid pJS3:(Y167I+R170L) as template. Theresulting PCR fragment were purified and the resulting PCR fragment wasused in a second PCR reaction as a primer with a second suitableopposite primer. This step was advantageous to be able to digest andclone the mutagenized region into the pJS3 shuttle vector.

[0445] Libraries of region 129-131, 132-133,134-135 have been preparedcontaining from 10,000 to 80,000 clones/library.

[0446] Ten randomly chosen colonies were sequenced to confirm themutations designed.

Example 5

[0447] Identification of Protease Variants with Increase AutoproteolyticActivity

[0448] The clones in each library constructed as described in example 4is tested for autoproteolytic stability as described above.

[0449] For each library 500 individual clones are incubated in amicrotiter plate overnight at 37° C. in 200 microliters LB medium with10 microg/ml Chloramphenicol (CAM).

[0450] With the SAVINASE® variant N:Y167I+R170L as reference strain twovariants with relative increased autoproteolytic stability wasidentified X:A133D+Y167I+R170L, Y: P129K+Y167I+R170L, andGG:P129K+P131H+Y167I+R170L.

[0451] This illustrates that the substitutions (P129K, P131H, A133D) inthe vicinity of the autoproteolytic cleavage site located between132-133 provide increased autoproteolytic stability.

Example 6

[0452] Comparative Fermentation Experiment with Variant(s) withIncreased Autoproteolytic Stability

[0453] The SAVINASE® variant “M: Y167I+R170L+A194P” was in afermentation experiment compared to its precursor variant “N:Y167I+R170L” not having the A194P substitution in the vicinity of theautoproteolytic split site located between residues 192-193.

[0454] Both variants were cloned in a pSX222 expression vectorbackground and fermented as described above in a 100 ml BPX mediumcontaining 10 microg/ml CAM.

[0455] After 5 days fermentation 1.5 ml of the BPX fermentation mediumwas centrifuged and the supernatant was used to measure the Proteolyticactivity (KPNU) as described above.

[0456] The fermentation medium containing the “M: Y167I+R170L+A194P”variant had a significant higher level of proteolytic activity ascompared to the fermentation medium containing the “N: Y167I+R170L”variant.

[0457] It is presently believed that both variants have the samespecific activity, and consequently it is presently believed that thehigher Proteolytic activity level in the fermentation medium containingthe “M: Y167I+R170L+A194P” variant is due to a relatively increasedautoproteolytic stability in the “M: Y167I+R170L+A194P” variant comparedto the “N: Y167I+R170L” precursor variant not having the A194Psubstitution.

[0458] Similar results were obtained with the variantsO:Y167A+R170S+A194P, P:Y167A+R170L+A194P, and Q:Y167A+R170N+A194Pcompared with their corresponding precursor variant without the A194Pmutation.

[0459] Further, similar results were obtained with the variant HH:A133P+Y167A+R170S compared with its corresponding precursor variantwithout the A133P mutation, showing that the substitution A133P giveincreased autoproteolytic activity.

1 35 1 49 PRT Bacillus amyloliquifaciens 1 Gly Gly Pro Ser Gly Ser AlaAla Leu Lys Ala Ala Val Asp Lys Ala 1 5 10 15 Val Ala Ser Glu Gly ThrSer Gly Ser Ser Ser Thr Val Gly Tyr Pro 20 25 30 Gly Lys Tyr Pro Phe SerSer Val Gly Pro Glu Leu Asp Val Met Ala 35 40 45 Pro 2 49 PRT Bacillussubtilis 2 Gly Gly Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Val Asp LysAla 1 5 10 15 Val Ser Ser Glu Gly Ser Ser Gly Ser Thr Ser Thr Val GlyTyr Pro 20 25 30 Ala Lys Tyr Pro Phe Ser Ser Ala Gly Ser Glu Leu Asp ValMet Ala 35 40 45 Pro 3 49 PRT Bacillus subtilis 3 Gly Gly Pro Ser GlySer Thr Ala Leu Lys Gln Ala Val Asp Lys Ala 1 5 10 15 Tyr Ala Ser SerGly Ser Ser Gly Ser Gln Asn Thr Ile Gly Tyr Pro 20 25 30 Ala Lys Tyr AspPhe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala 35 40 45 Pro 4 49 PRTBacillus licheniformis 4 Gly Gly Pro Ser Gly Ser Thr Ala Met Lys Gln AlaVal Asp Asn Ala 1 5 10 15 Tyr Ala Arg Ser Gly Ser Ser Gly Asn Thr AsnThr Ile Gly Tyr Pro 20 25 30 Ala Lys Tyr Asp Phe Ser Ser Val Gly Ala GluLeu Glu Val Met Ala 35 40 45 Pro 5 45 PRT Bacillus alcalohilus 5 Gly SerPro Ser Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala 1 5 10 15 ThrSer Arg Ser Gly Ala Gly Ser Ile Ser Tyr Pro Ala Arg Tyr Ala 20 25 30 PheSer Gln Tyr Gly Ala Gly Leu Asp Ile Val Ala Pro 35 40 45 6 45 PRTBacillus YaB 6 Gly Ser Ser Ala Gly Ser Ala Thr Met Glu Gln Ala Val AsnGln Ala 1 5 10 15 Thr Ala Ser Ser Gly Ala Gly Asn Val Gly Phe Pro AlaArg Tyr Ala 20 25 30 Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile Val Ala Pro35 40 45 7 45 PRT Bacillus lentus 7 Gly Ser Thr Ser Gly Ser Ser Thr LeuGlu Leu Ala Val Asn Arg Ala 1 5 10 15 Asn Asn Ala Thr Gly Arg Gln GlyVal Asn Tyr Pro Ala Arg Tyr Ser 20 25 30 Phe Ser Thr Tyr Gly Pro Glu IleGlu Ile Ser Ala Pro 35 40 45 8 46 PRT Bacillus subtilis 8 Gly Thr ThrSer Asp Ser Lys Ile Leu His Asp Ala Val Asn Lys Ala 1 5 10 15 Tyr GluGln Asp Gly Asn Gly Lys Pro Val Asn Tyr Pro Ala Ala Tyr 20 25 30 Ser PheSer Thr Thr Gly Asp Glu Val Glu Phe Ser Ala Pro 35 40 45 9 50 PRTThermoactinomyces vulgaris 9 Gly Gly Pro Ser Asp Val Pro Glu Leu Glu GluAla Val Lys Asn Ala 1 5 10 15 Val Lys Asn Glu Gly Asp Gly Asp Glu ArgThr Glu Glu Leu Ser Tyr 20 25 30 Pro Ala Ala Tyr Asn Phe Ser Asn Ala AsnLys Glu Ile Asp Leu Val 35 40 45 Ala Pro 50 10 45 PRT Bacillusamyloliquifaciens 10 Gly Gly Thr Val Gly Asn Ser Gly Leu Gln Gln Ala ValAsn Tyr Ala 1 5 10 15 Trp Asn Lys Ala Gly Asn Thr Ala Pro Asn Tyr ProAla Tyr Tyr Ser 20 25 30 Phe Ser Thr Tyr Gly Ser Trp Val Asp Val Ala AlaPro 35 40 45 11 47 PRT Dichelobacter nodosus 11 Gly Gly Gly Gly Gly CysSer Gln Asn Ser Gln Arg Met Ile Asp Lys 1 5 10 15 Thr Thr Asn Leu GluAsn Gln Asp Ala Ser Arg Thr Trp Pro Ser Ser 20 25 30 Cys Asn Phe Ser AsnTyr Gly Ala Arg Val His Leu Ala Ala Pro 35 40 45 12 47 PRT Xanthomonuscampestris 12 Gly Gly Gly Gly Ser Cys Ser Thr Thr Met Gln Asn Ala IleAsn Gly 1 5 10 15 Ala Val Ser Arg Asp Ala Ser Asn Val Ser Gly Ser LeuPro Ala Asn 20 25 30 Cys Ala Tyr Ser Asn Phe Gly Thr Gly Ile Asp Val SerAla Pro 35 40 45 13 58 PRT Bacillus subtilis 13 Gly Gly Gly Ser Gly LeuAsp Glu Trp Tyr Arg Asp Met Val Asn Ala 1 5 10 15 Trp Arg Ala Ala ThrAsp Leu Phe Ile Pro Gly Gly Pro Gly Ser Ile 20 25 30 Ala Asn Pro Ala AsnTyr Pro Phe Ser Leu Gln Gly Pro Ser Pro Tyr 35 40 45 Asp Glu Ile Lys ProGlu Ile Ser Ala Pro 50 55 14 59 PRT Enterococcus faecalis 14 Gly Ser TyrLys Asn Met Glu Ile Asp Asp Glu Arg Phe Thr Val Glu 1 5 10 15 Ala PheArg Lys Val Val Asn Tyr Ala Arg Lys Asn Glu Ser Arg Asp 20 25 30 Ile SerThr Gly Asn Glu Lys His Ile Pro Gly Gly Leu Glu Tyr Ser 35 40 45 Asn TyrGly Ser Asn Val Ser Ile Tyr Gly Pro 50 55 15 68 PRT Staphylococcusepidermis 15 Gly Asn Val Leu Ile Arg Asp Asp Glu Lys Val Asp Tyr Asp AlaLeu 1 5 10 15 Gly Lys Ala Ile Asn Tyr Ala Gln Lys Lys Asp Gly Ile AsnVal Lys 20 25 30 Lys Val Lys Glu Ile Asn Lys Lys Arg Thr Ser Lys Lys ValTyr Asp 35 40 45 Ser Pro Ala Asn Leu Asn Phe Ser Asn Tyr Gly Asn Asn PheIle Asp 50 55 60 Leu Met Thr Ile 65 16 71 PRT Streptococcus pyogenes 16Gly Asn Ala Ala Leu Ala Tyr Ala Asn Leu Pro Asp Glu Thr Lys Lys 1 5 1015 Ala Phe Asp Tyr Ala Lys Ser Lys Asp Ser Ser Phe Gly Gly Lys Thr 20 2530 Arg Leu Pro Leu Ala Asp His Pro Asp Tyr Gly Val Val Gly Thr Pro 35 4045 Ala Ala Ala Asp Phe Ser Ser Trp Gly Leu Thr Ala Asp Gly Asn Ile 50 5560 Lys Pro Asp Ile Ala Ala Pro 65 70 17 73 PRT Lactococcus lactis 17 GlySer Asn Ser Gly Asn Gln Thr Leu Glu Asp Pro Glu Leu Ala Ala 1 5 10 15Val Gln Asn Ala Asn Glu Ser Ser Gly Thr Ser Gly Ser Ala Thr Glu 20 25 30Gly Val Asn Lys Asp Tyr Tyr Gly Leu Gln Asp Asn Glu Met Val Gly 35 40 45Ser Pro Gly Thr Ser Arg Phe Thr Ser Tyr Gly Pro Val Ser Asn Leu 50 55 60Ser Thr Lys Pro Asp Ile Thr Ala Pro 65 70 18 63 PRT Serratia marcescens18 Gly Ile Ala Pro Asp Gln Pro Val Pro Thr Gly Gly His Ser Ala Met 1 510 15 Ser Thr Leu Leu Arg Ala Ala Arg His Tyr Asn Asn Tyr Asn Ile Pro 2025 30 Glu Ala Gln Lys Ser Leu Pro Tyr Ala Phe Pro Asp Val Leu Asn Ser 3540 45 Ser Thr Ser Cys Gly Gln Thr Ala Ser Tyr Cys Val Ser Ala Pro 50 5560 19 54 PRT Anabaena variabilis 19 Gly Gly Pro Asp Gly Lys Gln Lys ValPro Leu Pro Asp Ser Thr Arg 1 5 10 15 Leu Ala Met Asp Tyr Ala Ile AsnLys Gly Gly Asn Glu Ser Val Asp 20 25 30 Asn Asp Gly Tyr Ala Ser Tyr GluLys Tyr Ser Asp Phe Gly Thr Ala 35 40 45 Val Trp Cys Ala Phe Pro 50 2058 PRT Mus 20 Gly Pro Asn Asp Asp Gly Lys Thr Val Glu Gly Pro Gly ArgLeu Ala 1 5 10 15 Gln Lys Ala Phe Glu Tyr Gly Val Lys Gln Gly Gly GlyArg Gln Gly 20 25 30 Asp Asn Cys Asp Cys Asp Gly Tyr Thr Asp Ser Ile TyrTyr Ala Glu 35 40 45 Lys Cys Ser Ser Thr Leu Ala Thr Ser Tyr 50 55 21 57PRT Homo sapiens 21 Gly Thr Pro Asp Asn Gly Lys Thr Val Asp Gly Pro ArgAsp Val Thr 1 5 10 15 Leu Gln Ala Met Ala Asp Gly Val Asn Lys Gly GlyGly Ser Tyr Asp 20 25 30 Asp Cys Asn Cys Asp Gly Tyr Ala Ser Ser Met TrpTyr Asp Glu Ser 35 40 45 Cys Ser Ser Thr Leu Ala Ser Thr Phe 50 55 22 58PRT Homo sapiens 22 Gly Pro Glu Asp Asp Gly Lys Thr Val Asp Gly Pro AlaArg Leu Ala 1 5 10 15 Glu Glu Ala Phe Phe Arg Gly Val Ser Gln Gly GlyGly Arg Glu His 20 25 30 Asp Ser Cys Asn Cys Asp Gly Tyr Thr Asn Ser IleTyr Tyr Ser Glu 35 40 45 Ala Cys Ser Ser Thr Leu Ala Thr Thr Tyr 50 5523 58 PRT Drosophila melanogaster 23 Gly Pro Asp Asp Asp Gly Lys Thr ValAsp Gly Pro Gly Glu Leu Ala 1 5 10 15 Ser Arg Ala Phe Ile Glu Gly ThrThr Lys Gly Gly Gly Arg Glu Gln 20 25 30 Asp Asn Cys Asn Cys Asp Gly TyrThr Asn Ser Ile Trp Tyr Ser Glu 35 40 45 Lys Cys Ser Ser Thr Leu Ala ThrThr Tyr 50 55 24 58 PRT Kluyveromyces lactis 24 Gly Pro Ser Asp Asp GlyLys Thr Met Gln Ala Pro Asp Thr Leu Val 1 5 10 15 Lys Lys Ala Ile IleLys Gly Val Thr Glu Gly Gly Gly Met Phe Gly 20 25 30 Asp Ser Cys Asn PheAsp Gly Tyr Thr Asn Ser Ile Phe Tyr Ser Glu 35 40 45 Ser Cys Ser Ala ValMet Val Val Thr Tyr 50 55 25 58 PRT Saccharomyces cerevisiae 25 Gly ProAla Asp Asp Gly Arg His Leu Gln Gly Pro Ser Asp Leu Val 1 5 10 15 LysLys Ala Leu Val Lys Gly Val Thr Glu Gly Gly Gly Thr Arg Gly 20 25 30 AspAsn Cys Asn Tyr Asp Gly Tyr Thr Asn Ser Ile Tyr Tyr Ser Glu 35 40 45 GlyCys Ser Ala Val Met Ala Val Thr Tyr 50 55 26 45 PRT Vibrio alginolyticus26 Gly Gly Gly Gln Ser Val Ala Leu Asp Ser Ala Val Gln Ser Ala Val 1 510 15 Gln Ser Ser Asn Ala Asp Ala Cys Asn Tyr Ser Pro Ala Arg Val Ala 2025 30 Phe Ser Asn Trp Gly Ser Cys Val Asp Val Phe Ala Pro 35 40 45 27 45PRT Thermus rT41A 27 Gly Gly Gly Ala Ser Thr Ala Leu Asp Thr Ala Val MetAsn Ala Ile 1 5 10 15 Asn Ala Asp Asn Arg Asp Ala Cys Phe Tyr Ser ProAla Arg Val Thr 20 25 30 Phe Ser Asn Tyr Gly Arg Cys Leu Asp Leu Phe AlaPro 35 40 45 28 45 PRT Thermus aquaticus 28 Gly Gly Gly Val Ser Thr AlaLeu Asp Asn Ala Val Lys Asn Ser Ile 1 5 10 15 Ala Ala Asp Asn Ala AsnAla Cys Asn Tyr Ser Pro Ala Arg Val Ala 20 25 30 Phe Ser Asn Tyr Gly SerCys Val Asp Leu Phe Ala Pro 35 40 45 29 45 PRT Tritirachium album Limber29 Gly Gly Gly Tyr Ser Ser Ser Val Asn Ser Ala Ala Ala Arg Leu Gln 1 510 15 Ser Ser Asn Asn Ala Asp Ala Arg Asn Tyr Ser Pro Ala Ser Glu Pro 2025 30 Phe Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Gly Pro 35 40 45 30 45PRT Tritirachium album 30 Gly Gly Gly Tyr Ser Ser Ser Val Asn Ser AlaAla Ala Asn Leu Gln 1 5 10 15 Gln Ser Asn Asn Ala Asp Ala Arg Asn TyrSer Pro Ala Ser Glu Ser 20 25 30 Phe Ser Asn Tyr Gly Ser Val Leu Asp IlePhe Ala Pro 35 40 45 31 46 PRT Tritirachium album 31 Gly Gly Gly Pro SerSer Ser Ala Val Asn Arg Ala Ala Ala Glu Ile 1 5 10 15 Thr Ser Ala GluAla Thr Asp Ala Ser Ser Ser Ser Pro Ala Ser Glu 20 25 30 Glu Tyr Ser AsnPhe Gly Ser Val Val Asp Leu Leu Ala Pro 35 40 45 32 45 PRT Acremoniumchrysogenum 32 Gly Gly Gly Tyr Ser Ser Ala Phe Asn Asn Ala Val Asn ThrAla Tyr 1 5 10 15 Ser Arg Asp Asn Gln Asn Ala Ala Asn Tyr Ser Pro AlaSer Ala Ala 20 25 30 Phe Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Ala Pro35 40 45 33 45 PRT Aspergillus oryzae 33 Gly Gly Gly Tyr Ser Lys Ala PheAsn Asp Ala Val Glu Asn Ala Phe 1 5 10 15 Glu Gln Glu Asn Ser Asp AlaGly Gln Thr Ser Pro Ala Ser Ala Pro 20 25 30 Phe Ser Asn Phe Gly Lys ValVal Asp Val Phe Ala Pro 35 40 45 34 45 PRT Saccharomyces cerevisiae 34Gly Gly Gly Lys Ser Pro Ala Leu Asp Leu Ala Val Asn Ala Ala Val 1 5 1015 Glu Val Glu Asn Gln Asp Ala Cys Asn Thr Ser Pro Ala Ser Ala Asp 20 2530 Phe Ser Asn Trp Gly Lys Cys Val Asp Val Phe Ala Pro 35 40 45 35 51PRT Yarrowia lipolytica 35 Gly Gly Pro Lys Ser Ala Ser Gln Asp Ala LeuTrp Ser Arg Ala Thr 1 5 10 15 Gln Glu Asp Ala Val Asp Ala Cys Asn AspSer Pro Gly Asn Ile Gly 20 25 30 Gly Trp Ser Gly Gly Gln Gly Ser Asn TyrGly Thr Cys Val Asp Val 35 40 45 Phe Ala Pro 50

1. A modified subtilase comprising a mutation in an amino acid sequenceof a subtilase, wherein the mutation is a substitution, insertion ordeletion at one or more of positions 129, 130, 131, 132, 133, 134, 135,and 136, wherein each position is numbered according to the amino acidsequence of the mature subtilisin BPN′.
 2. The modified subtilase ofclaim 1, further comprising a mutation at one or more of positions 159,164, 165, 167, 170, and
 171. 3. A modified subtilase comprising amutation in an amino acid sequence of a subtilase, wherein the mutationis a substitution, insertion or deletion at one or more of positions189, 190, 191, 192, 193, and 196, wherein each position is numberedaccording to the amino acid sequence of the mature subtilisin BPN′.
 4. Amodified subtilase comprising a mutation in an amino acid sequence of asubtilase, wherein the mutation is a substitution, insertion or deletionat one or more of positions 129, 131, 136, 159, 164, 165, 167, 170 and171 and a substitution, insertion or deletion at one or more ofpositions 189, 190, 191, 192, 193, 194, 195 and 196, wherein eachposition is numbered according to the amino acid sequence of the maturesubtilisin BPN′.
 5. The modified subtilase of claim 1, comprising amutation at one or more of positions 129, 130, 131, 132, 133, 134, 135,and 136 and at one or more of positions 189, 190, 191, 192, 193, 194,195, and
 196. 6. The modified subtilase of claim 1, comprising one ormore of the following mutations: T129V, T129I, T129L, T129M, T129FA129V, A129I, A129L, A129M, A129F G131V, G131I, G131L, G131M, G131FK136V, K136I, K136L, K136M, K136F, S159V, S159I, S159L, S159M, S159F,T164V, T164I, T164L, T164M, T164F, K170V, K170I, K170L, K170M, K170F,Q136V, Q136I, Q136L, Q136M, Q136F, T159V, T159I, T159L, T159M, T159F,A164V, A164I, A164L, A164M, A164F, Y170V, Y170I, Y170L, Y170M, Y170F,Y171A, Y171H, Y171N, Y171P, Y171C, Y171W, Y171Q, Y171S, Y171T, Y171G,Y171V, Y171I, Y171L, Y171M, Y171F, E136V, E136I, E136L, E136M, E136F,G159V, G159I, G159L, G159M, G159F, G164V, G164I, G164L, G164M, G164F,S164V, S164I, S164L, S164M, S164F, Y167A, Y167H, Y167N, Y167P, Y167C,Y167W, Y167Q, Y167S, Y167T, Y167G, Y167V, Y167I, Y167L, Y167M, Y167F,R170A, R170H, R170N, R170P, R170C, R170W, R170Q, R170S, R170T, R170Y,R170G, and R170V, R170I, R170L, R170M, R170F.
 7. The modified subtilaseof claim 1, comprising one or more of the following mutations: T129V,T129I, T129L, T129M, T129F, A129V, A129I, A129L, A129M, A129F, G131V,G131I, G131L, G131M, G131F, K136V, K136I, K136L, K136M, K136F, S159V,S159I, S159L, S159M, S159F, T164V, T164I, T164L, T164M, T164F, K170V,K170I, K170L, K170M, K170F, Q136V, Q136I, Q136L, Q136M, Q136F, T159V,T159I, T159L, T159M, T159F, A164V, A164I, A164L, A164M, A164F, Y170V,Y170I, Y170L, Y170M, Y170F, Y171A, Y171H, Y171N, Y171P, Y171C, Y171W,Y171Y171Q, Y171S, Y171T, Y171G, Y171V, Y171I, Y171L, Y171M, Y171F,E136V, E136I, E136L, E136M, E136F, G159V, G159I, G159L, G159M, G159F,G164V, G164I, G164L, G164M, G164F, S164V, S164I, S164L, S164M, S164F,Y167A, Y167H, Y167N, Y167P, Y167C, Y167W, Y167Q, Y167S, Y167T, Y167G,Y167V, Y167I, Y167L, Y167M, Y167F, R170A, R170H, R170N, R170P, R170C,R170W, R170Q, R170S, R170T, R170Y, R170G, and R170V, R170I, R170L,R170M, R170F.
 8. The modified subtilase of claim 1, comprising one ormore of the following mutations: T129V, T129I, T129L, T129M, T129F,A129V, A129I, A129L, A129M, A129F, G131V, G131I, G131L, G131M, G131F,K136V, K136I, K136L, K136M, K136F, S159V, S159I, S159L, S159M, S159F,T164V, T164I, T164L, T164M, T164F, K170V, K170I, K170L, K170M, K170F,Q136V, Q136I, Q136L, Q136M, Q136F, T159V, T159I, T159L, T159M, T159F,A164V, A164I, A164L, A164M, A164F, Y170V, Y170I, Y170L, Y170M, Y170F,Y171A, Y171H, Y171N, Y171P, Y171C, Y171W, Y171Q, Y171S, Y171T, Y171G,Y171V, Y171I, Y171L, Y171M, Y171F E136V, E136I, E136L, E136M, E136F,G159V, G159I, G159L, G159M, G159F, G164V, G164I, G164L, G164M, G164F,S164V, S164I, S164L, S164M, S164F, Y167A, Y167H, Y167N, Y167P, Y167C,Y167W, Y167Q, Y167S, Y167T, Y167G Y167V, Y167I, Y167L, Y167M, Y167F,R170A, R170H, R170N, R170P, R170C, R170W, R170Q, R170S, R170T, R170Y,R170G, and R170V, R170I, R170L, R170M, R170F; in combination with amodification in one or more of any of the following positions: 130, 131,132, 133, 134, 135, 136, and 190, 191, 192, 193, 194, 195,
 196. 9. Themodified subtilase of claim 1, wherein the subtilase is a sub-group I-S1subtilase.
 10. The modified subtilase of claim 9, wherein the subtilaseis subtilisin I168, subtilisin BPN′, subtilisin DY, or subtilisinCarlsberg.
 11. The modified subtilase of claim 1, wherein the subtilaseis a sub-group I-S2 subtilase.
 12. The modified subtilase of claim 11,wherein the subtilase is subtilisin 147, subtilisin 309, subtilisinPB92, or subtilisin YaB.
 13. The modified subtilase of claim 11, whereinthe subtilase is thermitase.
 14. The modified subtilase of claim 1,comprising at least one further mutation at one or more other positions,wherein said further mutation is selected from the group consisting of asubstitution, insertion or deletion.
 15. The modified subtilase of claim14, wherein the one or more other positions are selected from the groupconsisting of: 27, 36, 57, 76, 97, 101, 104, 120, 123, 206, 218, 222,224, 235 and
 274. 16. The modified subtilase of claim 15, wherein thesubtilase is an I-S2 sub-group subtilase and the at least one furthermutation is selected from the group consisting of K27R, *36D, S57P,N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, Q206E, N218S,M222S, M222A, T224S, K235L, and T274A.
 17. The modified subtilase ofclaim 17 wherein the one at least one further mutation is selected fromthe group consisting of V104N+S101G, K27R+V104Y+N123S+T274A, orN76D+V104A, and any other combination of K27R, N76D, S101G, V104A,V104N, V104Y, N123S, and T274A.
 18. The modified subtilase of claim 1,wherein the mutation is selected from the group consisting of: A:Y167I + R070L + A133P B: Y167I + R170L + T134P C: Y167I + R170L +A133P + T134P D: Y167I + R170L + V104C + S132C E: Y167I + R170L +A108C + T134C F: Y167A + R170S + F189A G: Y167A + R170S + Y192A H:Y167A + R170S + Y192P I: Y167A + R170S + Y192A + A194P J: Y167A +R170S + Y192P + A194P K: Y167A + R170S + F189G L: Y167A + R170S + F189EM: Y167A + R170S + F189R N: Y1671 + R170L M: Y1671 + R170L + A194P O:Y167A + R170S + A194P P: Y167A + R170L + A194P Q: Y167A + R170N + A194PR: V104C + S132C + Y1671 + R170L S: A108C + T134C + Y1671 + R170L T:V104C + S132C + Y167A + R170S U: V104C + S132C + Y167A + R170L V:V104C + S132C + Y167A + R170N X: A133D + Y1671 + R170L Y: P129K +Y1671 + R170L Z: A133P + Y167A + R170S + A194P AA: T134P + Y167A +R170S + A194P BB: A133P + T134P + Y167A + R170S + A194P CC: A133P +Y167A + R170N + A194P DD: T134P + Y167A + R170N + A194P EE: A133P +T134P + Y167A + R170N + A194P FF: A133P + Y167A + R170L GG: P129K +P131H + Y1671 + R170L HH: A133P + Y167A + R170S II: A133P + Y167A +R170N JJ: Y167A + R170S + F189K and KK: V104C + T134C + Y167A + R170S.


19. The modified subtilase of claim 1, wherein the mutation comprisesone or both of A194P and A133P; and further comprises a mutationselected from the group consisting of R170A R170C R170F R170G R170HR170I R170L R170N R170M R170P R170Q R170S R170T R170V R170Y Y167A +R170A Y167C + R170A Y167F + R170A Y167G + R170A Y167H + R170A Y167I +R170A Y167L + R170A Y167M + R170A Y167N + R170A Y167P + RI70A Y167Q +R170A Y167S + R170A Y167T + RI70A Y167V + R170A Y167A + R170C Y167C +R170C Y167F + R170C Y167G + R170C Y167H + R170C Y167I + R170C Y167L +R170C Y167M + R170C Y167N + R170C Y167P + R170C Y167Q + R170C Y167S +R170C Y167T + R170C Y167V + R170C Y167A + R170F Y167C + R170F Y167F +R170F Y167G + R170F Y167H + R170F Y167I + R170F Y167L + R170F Y167M +R170F Y167N + R170F Y167P + R170F Y167Q + R170F Y167S + R170P Y167T +R170P Y167V + R170P Y167A + R170Q Y167C + R170Q Y167F + R170Q Y167G +R170Q Y167H + R170Q Y167I + R170Q Y167L + R170Q Y167M + R170Q Y167N +R170Q Y167P + R170Q Y167Q + R170Q Y167S + R170Q Y167T + R170Q Y167V +R170Q Y167A + R170S Y167C + R170S Y167F + R170S Y167G + R170S Y167H +R170S Y1671 + R170S Y167L + R170S Y167M + R170S Y167N + R170S Y167P +R170S Y167Q + R170S Y167S + R170S Y167T + R170S Y167V + R17QS Y167A +R170T Y167C + R170T Y167F + R170T Y167G + R170T Y167H + R170T Y1671 +R170T Y167L + R170T Y167M + R170T Y167N + R170T Y167P + R170T Y167Q +R170T Y167S + R170T Y167T + R170T Y167V + R170T Y167A + R170V Y167C +R170V Y167F + R170V Y167G + R170V Y167H + R170V Y167I + R17QV Y167L +R170V Y167M + R170V Y167N + R170V Y167P + R170V Y167Q + R170V Y167S +R170V Y167T + R17QV Y167V + R170V Y167A + R170Y Y167C + R170Y Y167F +R170Y Y167G + R170Y Y167H + R170Y Y167I + R170Y Y167L + R17QY Y167M +R170Y Y167N + R170Y Y167P + R170Y Y167Q + R170Y Y167S + R170Y Y167T +R170Y and Y167V + R170Y.


20. A composition comprising a modified subtilase of claim 1 and asurfactant.
 21. The composition of claim 20, which additionallycomprises an amylase, cellulase, cutinase, lipase, oxidoreductase, oranother protease.
 22. A DNA sequence encoding a modified subtilase ofclaim
 1. 23. A vector comprising a DNA sequence of claim
 22. 24. Amicrobial host cell transformed with a vector of claim
 23. 25. Themicrobial host of claim 24, which is a bacterium
 26. The microbial hostof claim 25, which is a Bacillus cell.
 27. The microbial host of claim26, which is a B. lentus cell.
 28. The microbial host of claim 24, whichis a fungus or yeast.
 29. The microbial host of claim 28, which is afilamentous fungus.
 30. The microbial host of claim 29, which is anAspergillus cell.
 31. A method for producing a modified subtilase,comprising (a) culturing a host of claim 24 under conditions conduciveto the expression and secretion of the modified subtilase, and (b)recovering the modified subtilase.
 32. A method for the identificationof a protease variant exhibiting autoproteolytic stability, whichcomprises effecting a mutation in DNA encoding a subtilase enzyme or itspre- or preproenzyme at one or more of the positions corresponding toamino acid P129, P131, E136, G159, S164, I165, Y167, R170, Y171 (insubtilisin BPN′ numbering); (a) transforming a Bacillus strain with saidmutated DNA; (b) selecting strains producing such protease variants; (c)fermenting/growing such a strain; (d) recovering said protease variant,and (e) testing for improved storage stability and/or wash performancein detergents.